Assessment of Biocompatibility of 16HBE14o- Human Bronchial Epithelial Cells in Alginate-Methylcellulose Bioinks Revealed Spheroid Formation

Assessment of Biocompatibility of 16HBE14o- Human Bronchial Epithelial Cells in Alginate-Methylcellulose Bioinks Revealed Spheroid Formation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Assessment of Biocompatibility of 16HBE14o- Human Bronchial Epithelial Cells in Alginate-Methylcellulose Bioinks Revealed Spheroid Formation Nathan Wood, Hongmin Qin, Wanhe Li, Esther Doria This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4784339/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The biocompatibility of 16HBE14o- human bronchial epithelial cells in ionically cross-linked alginate-methylcellulose bioinks was assessed. This was accomplished by encapsulating 16HBE14o- cells in either a sodium alginate bioink or a bioink with sodium alginate and added methylcellulose in a 1:1 ratio. To differentiate the effects of methylcellulose from those of cross-linking on cell viability, two concentrations of calcium chloride cross-linker were used for both alginate only and alginate-methylcellulose bioinks. Using fluorescence microscopy, it was observed that bioinks with methylcellulose showed a small but significant reduced cell viability and a decreased presence of cell spheroids compared to their methylcellulose free alginate counterparts. However, alginate-methylcellulose bioinks still supported cell proliferation and appeared to be biocompatible. Additionally, the concentration of cross-linker seemed to impact cell viability. This study has implications for the use of methylcellulose as a viscosity tuner for both general 3D 16HBE14o- human epithelial cell culture and 3D bioprinting. The presence of spheroids suggests that alginate-methylcellulose bioinks could be useful in generating 3D 16HBE14o- human epithelial cell culture to address questions in cell biology, including signal transduction, metabolic activity, and cancer hallmarks. Biological sciences/Biological techniques/Cytological techniques/Cell culture Biological sciences/Biological techniques/Biological models/Respiratory system models 3D spheroid epithelium bioink alginate methylcellulose Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction 3D cell culture is used to address life science questions by growing cells in a 3D spatial orientation that more closely mimics their in vivo environment [ 1 ] . This approach allows for a more physiologically sound understanding of interactions with the cell membrane and cytoskeleton, extracellular matrix, and signal transduction. It also addresses practical problems related to tissue grafting, drug discovery, and drug delivery [ 1 , 2 ] . Of particular interest is the formation of cell spheroids, which are 3D cell aggregates that better mimic tissues and tumors. Unlike 2D monolayers, which are forced to adhere to a very stiff plastic or glass surface with mostly uniform exposure to metabolites and oxygen, spheroids experience metabolic gradients such as localized hypoxia, allowing for more realistic drug screenings and stress studies [ 2 , 3 ] . 3D bioprinting can enhance the scalability and complexity of current 3D cell culturing methods [ 4 ] . Extrusion-based bioprinting often employs mechanical pressure to deposit droplets or filaments consisting of a biomaterial, referred to as a bioink, onto a provided surface in a desired pattern [ 5 ] . Cell culturing media, in its typically used state, is unsuitable for 3D bioprinting. Therefore, it is of interest to produce materials that are viscous enough for adequate printing, while also posing minimal hazard to the cells [ 6 ] . Derived from brown algae, sodium alginate and its modified conjugates are popular base components for bioinks due to its low cost, biocompatibility both in vitro and in vivo, and ability to cross-link as a hydrogel when exposed to cations such as calcium [ 7 , 8 ] . To enhance the physical and chemical properties of the bioink and its resulting hydrogel, and to meet the physiological needs for cells interacting with the hydrogel, additional materials, such as extracellular matrix components and cellulose derivatives are incorporated. Extracellular matrix (ECM) components such as collagen and fibronectin are essential for epithelial cells, which interact with ECM via membrane integrin receptors. The absence of ECM can lead to a form of programmed death referred to as anoikis [ 9 – 11 ] . To prevent this, bioinks often include ECM components or materials modified to have peptide motifs that serve as integrin ligands [ 12 , 13 ] . Soluble cellulose derivatives such as methylcellulose are commonly added to modify the viscosity of the bioink before cross-linking, and to modify the stiffness and compression behaviors of the hydrogel after cross-linking [ 14 ] . Previous report by Li et al. evaluated the rheological characteristics of alginate-methylcellulose mixtures at ratios of 1:3, 3:3, and 3:9 (% w/v) for extrusion bioprinting, observing greater than 95% cell viability when L929 mouse fibroblast cells were incorporated between layers of the alginate-methylcellulose bioink [ 15 ] . Duin et al. successfully printed a 3:9 (%) alginate-methylcellulose hydrogel laden with mouse pancreatic islets, achieving 60–80% cell viability over seven days [ 16 ] . Ahlfield et al. demonstrated the use of an alginate-methylcellulose mixture dissolved in fresh frozen human plasma to culture mesenchymal stem cells, human dental pulp cells, and human umbilical vein endothelial cells, and human preosteoclast cells [ 17 ] . Cells forming spheroids in 3D culture replicate in vivo conditions, crucial for studying disease mechanisms and drug responses with greater accuracy in biomedical research and regenerative medicine. Regarding the 3D culturing of lung cells, Celis et al. successfully formed spheroids of 16HBE14o- human bronchial epithelial cells using a hanging drop method. They demonstrated that cells in the spheroids experience oxidative stress and transcriptional shifts concerning cell-cell interactions [ 18 ] . To study the effect of diesel exhaust particles on Transforming Growth Factor Beta (TGF-β) and markers of the Epithelium to Mesenchymal Transition (EMT), Baarsma et al. employed a magnetic bioprinting method in which BEAS-2B human bronchial epithelial cells were cultured with proprietary NanoShuttle™ particles, allowing cells form spheroids by placing a magnet underneath the culture vessel [ 19 ] . Regarding the use of methylcellulose, Tam had employed furan-modified hyaluronic acid combined with thiolated methylcellulose to study the invasive properties of stem cell derived smooth muscle cells with loss of function in tuberous sclerosis complex 1 or 2, as a model for lymphangioleimyomatosis [ 20 ] . Using a fibrous scaffold derived via the electrospinning of poly(ε-caprolactone) and methylcellulose, Gonçalves reported that while proliferation of 16HBE human bronchial epithelial cells are observed between 1 to 7 days, proliferation collapses between 7 and 14 days [ 21 ] . The study assessed the biocompatibility of methylcellulose as an alginate bioink additive for 3D culturing, and potentially3D bioprinting, of 16HBE14o- human bronchial epithelial cells. It compared cell viability and spheroid formation in bioinks containing only ionically cross-linked sodium alginate, as well as hydrogels bearing sodium alginate and methylcellulose in a 1:1 ratio (test groups shown in Table 1 ). The study found that while alginate-methylcellulose bioinks cross-linked with 300 mM calcium chloride had the lowest initial cell viability, these bioinks supported greater spheroid cell proliferation over nine days, suggesting their potential for 3D culturing. This work provides preliminary insights into using alginate-methylcellulose bioinks for 3D bioprinting, enhancing complex 3D culturing techniques. Table 1 Composition of Bioinks Ingredient Bioink 1 Bioink 2 Bioink 3 Bioink 4 Sodium Alginate + + + + ECM Components + + + + 16HBE14o- + + + + Methylcellulose + + - - 100 mM CaCl 2 - + - + 300 mM CaCl 2 + - + - Results Cell Viability in Bioinks The impact of the addition of methylcellulose and concentration of cross-linking agent on cell viability was evaluated by distinguishing living cells that fluoresce exclusively blue from dead cells that fluoresce both blue and green within each bioink (Fig. 2 A). All four bioinks maintained cell viability over 3, 6, and 9 days, indicating that methylcellulose is not cytotoxic to 16HBE14o- cells (Fig. 2 B and Table 2 ). On Day 3, all bioinks exhibited the lowest viability. By Day 6, the bioink without methylcellulose cross-linked with 300 mM CaCl 2 achieved the highest observed viability at 81.68%. Bioinks containing methylcellulose cross-linked with 300 mM CaCl 2 consistently showed the lowest viability across all days (Day 3 at 67.09%, 6 at 62.62%), and 9 at 67.76%). Table 2 Cell Viabilities in Bioink for 3, 6, and 9 Days. All units are percentages of living cells relative to the total cells. IQR Represents the interquartile range. Day 3 4% Methylcellulose No Methylcellulose 100 mM CaCl 2 300 mM CaCl 2 100 mM CaCl 2 300 mM CaCl 2 Mean (%) 58.44 67.09 68.08 64.82 Median (%) 66.67 57.14 66.67 65.83 Standard Deviation ± 18.18 ± 15.49 ± 9.2 ± 7.85 IQR 22.97 16.67 13.96 10.49 Day 6 Mean (%) 75.56 62.62 80.55 81.68 Median (%) 70.83 61.82 79.29 82.84 Standard Deviation ± 14.1 ± 15.11 ± 9.25 ± 10.66 IQR 17.26 17.20 14.9 12.22 Day 9 Mean (%) 62.17 67.26 74.27 76.12 Median (%) 70.0 66.67 77.35 78.79 Standard Deviation ± 8.87 ± 16.31 ± 12.61 ± 27.45 IQR 32.2 16.29 12.22 16.12 In bioinks without methylcellulose, no statistical difference was observed in cell viability between those cross-linked with 100 mM or 300 mM CaCl2 solutions on any examined days. However, bioinks containing methylcellulose and cross-linked with 300 mM CaCl2 exhibited lower viability compared to those cross-linked with 100 mM CaCl2 on Day 3 ( p = 0.0096 ), but not on Day 6 ( p = 0.1412 ) or Day 9 ( p = 1.0 ). Overall, these results indicate that all four bioinks are biocompatible to 16HBE14o human bronchial epithelial cells. However, the addition of 4% methylcellulose to alginate hydrogels reduces the viability of encapsulated human bronchial epithelial cells compared to hydrogels without methylcellulose, with this reduced viability exacerbated by high concentrations of CaCl 2 cross-linker. Formation of Spheroids in Bioinks On Day 3, bioinks containing methylcellulose primarily exhibited single cells and small cell spheroids containing 2–4 cells. Notable exceptions were a single 13–24 cell spheroid in Bioink 1 and a single 5–8 cell and 9–12 cell spheroid in Bioink 2 (Fig. 3 B, Table 3 ). In contrast, bioinks without methylcellulose already showed a significant proportion of spheroids bearing 5–8 cells by day 3. By Day 6, spheroids containing 5–8 cells became the most common category across all bioinks. Spheroids with 9–12, 13–24, and more than 24 cells began to appear more frequently in bioinks without methylcellulose. By Day 9, spheroids with 13–24 cells became the most prevalent group across all bioinks. These observations suggested that adding methylcellulose to an alginate bioink might have initially delayed the proliferation of human bronchial epithelial cells. However, this delay was overcome by Day 6. The formation of spheroids in all bioinks demonstrated their biocompatibility for the 16HBE14o- cells. Table 3 Distributions of cell clusters in bioinks on days 3 to 9 based on cell count. Day 3 Types of cell clusters 4% Methylcellulose No Methylcellulose 100 mM CaCl 2 300 mM CaCl 2 100 mM CaCl 2 300 mM CaCl 2 1 Cell 3 7 3 9 2–4 Cells 10 11 14 17 5–8 Cells 0 1 13 7 9–12 Cells 0 1 2 2 13–24 Cells 1 0 2 1 > 24 Cells 0 0 0 0 Day 6 1 Cell 5 3 1 5 2–4 Cells 4 4 10 4 5–8 Cells 7 7 11 12 9–12 Cells 3 2 9 7 13–24 Cells 2 3 9 7 > 24 Cells 0 0 2 0 Day 9 1 Cell 0 1 1 1 2–4 Cells 1 2 4 4 5–8 Cells 5 7 8 5 9–12 Cells 1 1 1 3 13–24 Cells 7 5 7 14 > 24 Cells 1 2 8 8 Spatial Confinement of Cells in Spheroids To analyze the spatial confinement of cells within spheroid, their volumes were estimated using the spheroid’s area under maximum intensity projection and the product of confocal Z stacks and the acquisition’s Nyquist Rate, then divided by the manually counted number of cells per spheroid. Spheroids containing smaller number of cells (2–4 cells, 5–8 cells, and 9–12 cells) exhibited varying spatial confinement, excluding outliers, ranging approximately from 419.17 to 5728.82 µm³cell − 1 (Fig. 4 A-C). Spheroids with 13–24 cells and greater than 24 cells appeared less spatially confined, with volume per cell ranges from 1855.95 to 8649.05 and 2562.68 to 9402.33 µm³cell − 1 , respectively (Fig. 4 D-E). The addition of methylcellulose into an alginate bioink has minimal impact on the spatial confinement of 16HBE14o- cells within a spheroid. Across 3, 6, and 9 days, all four bioinks showed consistent spatial confinement of cells within spheroids of the same cell count category (Fig. 3 A-E). An exception to this were Day 3 spheroids with 2–4 cells in bioinks with methylcellulose cross-linked with 300 mM CaCl 2 , which were less spatially confined than those in bioinks with methylcellulose cross-linked with 100 mM CaCl 2 ( p = 0.0445) ; however, this difference were not preserved on Day 6 ( p = 1.0 ). Additionally, the concentration of CaCl 2 cross-linker had negligible effect on the spatial confinement of 16HBE14o- cells, despite a previous report indicating its influence on alginate hydrogel swelling [ 22 ] . Discussion This study reports the growth of human bronchial epithelial 16HBE14o- cells into spheroids using bioinks containing alginate and methylcellulose in a 1:1 ratio. All four bioinks demonstrated suitable biocompatibility with this cell line, as accessed by cell viability and cell proliferation. Small but statistically significant effects were observed when bioinks were added with methylcellulose, which exhibited decreased cell viability (Fig. 2 B) and a slightly impaired cell proliferation rate (Figs. 2 – 3 and Tables 2 – 3 ). Methylcellulose serves as a viscosity modifier before cross-linking and affects stiffness and porosity after cross-linking, making it a valuable tool in 3D bioprinting for enhancing the scalability and complexity of culturing human bronchial epithelial spheroids. Future pursuits will likely explore the use of spheroids in bioinks to examine markers for epithelial to mesenchymal transition, a cancer hallmark in which epithelial cells degenerate into a multipotent and invasive state [ 19 , 23 ] . Additionally, pulmonary fibrosis, characterized by excessive stiffening of extracellular matrix may be simulated by increasing or decreasing bioink stiffness with different concentrations of methylcellulose and ECM components [ 24 – 26 ] . This study demonstrates the feasibility of using custom alginate and methylcellulose bioinks for 3D culture of 16HBE14o- cells, potentially mimicking complex in vitro physiological conditions. Materials and Methods Cell Culture The 16HBE14o- (SCC150),an immortalized line of human bronchial epithelial cells, was purchased from Millipore Sigma in January 2024. Maintained in a seed-stock system, cells used in this study were used for no more than five passages since receipt from Millipore. Cells prior to mixing with bioinks were cultured in Alpha Modified Eagle Medium (α-MEM, Sigma Aldrich) supplemented with Fetal Bovine Serum (10% v/v, Corning 35-010-CV Lot 27823001) and L-Glutamine (2 mM). Cells tested negative for mycoplasma contamination via confirming a lack of extranuclear Hoescht 33342 DNA fluorescence on a subsample of cells grown as a monolayer. Antibiotic-antimycotic mixture (100 U mL − 1 penicillin, 100 µg mL − 1 streptomycin, 0.5 µg mL − 1 amphotericin B) was not used until cells were mixed with bioinks. All cell culture was in a humidified 37°C incubator with 5 percent CO 2 supply, with media exchange performed every 48 hours. Preparation of Extracellular Matrix Components Extracellular Matrix (ECM) components were provided by the preparation of a tissue culture flask coating solution per Millipore Sigma protocols, using Fatty Acid Free Bovine Serum Albumin (100 µg mL − 1 , Cat: 126575), Advanced Biomatrix PureCol Type 1 Bovine collagen with trace Type 3 collagen (30 µg mL − 1 , Cat: 5006), and Human Plasma Fibronectin (10 µg mL − 1 , Cat F2006). Preparation of Alginate-Methylcellulose and Alginate Bioinks Alginate and methylcellulose powders were weighed separately, then mixed. The powders were then double wrapped in aluminum foil and placed in a metal tin with folded aluminum foil to separate the wrapped powders from the bottom of the tin. The tin was then covered and placed on a hot plate set at approximately 80°C (Fig. 1A). After two hours, the tin was allowed to cool to room temperature without intervention. After this, the powdered alginate and methylcellulose mixture was opened in a disinfected biosafety hood and exposed to the hood’s UV lamp for 15 minutes [28] (Fig. 1B). The flask coating solution outlined in Section 2.2.1 was added to sterile flasks with magnetic stir bars pre-cooled to 4°C using a serological pipette and was supplemented with antibiotic-antimycotic mixture to the concentration specified in Section 2.1. A magnetic stir plate was placed inside the biosafety hood, and the two solutions were mixed by slowly pouring in the alginate-methylcellulose mix. Once stirred until no visible particles were observed, the solutions were then stirred on the same stir plate but in a 4°C cold room for 36 hours. As the solutions mix, the viscosity will increase, and the qualitative stir power of the plates will need to be increased. This process was also done for a bioink with only sodium alginate (4% w/v) to serve as a control for the alginate-methylcellulose bioink. Cells grown to 80% confluence were loosened using Trypsin-EDTA (0.5 g L − 1 Trypsin, 0.2 g L − 1 EDTA) solution for 6 minutes. Cell counts were determined using a Nexcelom Cellometer Auto T4 and its proprietary two-chamber slide. 1 mL resuspended cells were added to 2 mL bioink and mixed thoroughly using a glass stir rod. The final concentration of the cell-laden bioink was 2.06x10 5 cell mL − 1 . Cell-laden inks without methylcellulose were pipetted in 100 µL volumes into wells of a six well plate, each containing a glass coverslip, while cell-laden inks with methylcellulose were transferred onto glass coverslips in wells using a metal scoopula. All inks were cross-linked in 500 µL of either 100 mM or 300 mM CaCl 2 solution in water. Cross-linking was done in a delayed sequence such that no individual bioink was bathed for more than 5 minutes. All experimental groups were done in triplicate. Data and Image Analysis Images were processed using a Fiji is Just ImageJ (FIJI) version 2.15.1. Any observations of drift along the Z-stack were corrected by the Correct_3d_Drift.py script provided as an intrinsic FIJI plugin. Linear adjustments to brightness and contrast were performed with GIMP 2.10.34. Statistical analysis was conducted using LibreOffice Calc 7.4.7.2 and Rstudio version 2023.12.1 + 402 with R version 4.2.2 Patch r83330. Levene’s Test is provided as part of the R package car 3.1-2. Dunn’s Post-Hoc Test is provided as part of R package dunn.test 1.3.2. Visualization was accomplished using ggplot2 3.5.1 and ggsci 3.1.0. Evaluating Cell Viability Using Epifluorescent Microscopy Cell-laden bioinks, cross-linked and incubating in culture media, were treated with Invitrogen ReadyProbes Blue/Green Viability Imaging Kit (R37609, Thermo Fisher), containing Hoescht 33342 to stain all cells, and NucGreen Dead, which only permeates the compromised membranes of dead or dying cells. Two drops of each solution were added to each well. Living cells should exclusively fluoresce blue, whereas dead and dying cells should fluoresce green and blue (Fig. 2A). Cells were then incubated for 30 minutes. Cells were imaged using an Echo Revolution microscope in its inverted configuration, with an incubator chamber mated to the microscope set to 37°C, and given a 5 percent CO 2 , 20 percent O 2 mixture with 150 cc flow rate. Images were taken using a dry infinity corrected Olympus PlanLUC FLN 10X/0.3 using Chroma DAPI and FITC filters and a monochrome sCMOS detector and acquisition software (version 2.0.0.0) intrinsic to the microscope. For analysis of cell viability, the Kruskal-Wallis statistical test followed Dunn’s post-hoc test with comparisons adjusted using Bonferroni correction. P values below 0.05 were considered significant. The Kruskal-Wallis test was chosen as the distribution of residuals for test groups did not follow a normal distribution both graphically and per the Shapiro-Wilk’s Test (lowest p = 0.000395 ), while maintaining homogeny of variance using Levene’s Test (lowest p = 0.0829 ). On plots, statistical equivalence is abbreviated as Not Significant (N.S), whereas significance is abbreviated as * ( p < 0.05 ), ** ( p < 0.01 ), or *** ( p < 0.001 ). Estimating Number of Cells in Spheroids Samples were washed three times with 1X Phosphate Buffered Saline, then fixed with paraformaldehyde in 1X PBS solution (4% w/v) for 2 hours. Samples were then washed three times again with 1X PBS then treated with SYTOX Green nucleic acid stain (1:2000 dilution, Thermo Fisher) to stain cell nuclei for 30 minutes in the dark. Samples transferred to a microscope slide and treated with Invitrogen SlowFade glycerol mountant. Coverslips were placed on top of the bioink samples prior to imaging with a Zeiss Examiner.Z1 with LSM900 Laser Scanning Confocal microscope, in frame scanning mode with 488 nm excitation laser and a Zeiss Plan Apochromat 20X/0.8 dry objective. When examining the number of cells within an spheroid, the approximate number of cells was counted manually and then each respective cell count was organized into categories of 1, 2–4, 5–8, 9–12, 13–24, and greater than 24 cells, visualized in Fig. 3A and quantified in Table 3. Estimating Volume of Spheroids To examine if the addition of methylcellulose or different concentrations of cross-linking agents impacts how spatially confined cells are within spheroids, the approximate volume of observed spheroids was divided by the number of cells found in the particular spheroid. Axial height of a spheroid was approximated using Z stacks. Lower and upper Z sections were selected from when a given spheroid becomes totally out of focus, with the difference begin multiplied with the acquisition’s Nyquist Rate (0.53 µm). Using the area determined from the maximum intensity projection of the Z stack and the axial height, the volume of a spheroid was calculated as follows. $$\:V=\frac{4}{3}\pi\:{r}^{2}c$$ 1 Where: - \(\:\pi\:{r}^{2}\) represents the area determined from a maximum intensity projection - \(\:c\) is the axial radius, or half of the axial height, as calculated from upper and lower Z stacks The Kruskal Wallis Test with Dunn’s post-hoc test with Bonferroni correction was used to compare units volume per cell. Groups in which there were less than four observations were discarded from future analysis. This decision was made as the distribution data (spheroidal volume per cell) appears to not be normal per Shapiro-Wilk test (lowest p = 1.026x10 − 5 ), while still maintaining homogeny of variance per Levene’s Test (lowest p = 0.1011 , groups below 0.05 had sample sizes less than four and were discarded). Statistical significance on plots is designated in the same manner for cell viability in Section 2.3.1. Data Release Statement Data used in this report, including image data, statistical reports, and tabulated data can be provided upon reasonable request by the corresponding author. Declarations Competing Interests Statement The authors have no competing interests to report. Author Contribution N.W conceived the experiments, prepared bioinks, lead image acquisition, and analyzed data. E.D. assisted with confocal microscopy. W.L. provided the Zeiss Examiner.Z1 and LSM 900 Unit. Z.P. is the co-principal investigator, provided research advisory, and reviewed the manuscript. H.Q. is the principal investigator, provided research advisory, and reviewed the manuscript. All authors have received a copy of the manuscript. Acknowledgement This research was funded by the U.S. Air Force Office of Scientific Research (AFOSR), grant number FA9550-23-1-0599, and FA9550-23-1-0156. 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Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes. Journal of Materials Science: Materials in Medicine 30, (2019). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4784339","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":341865452,"identity":"8e5207df-50d5-4ac5-a2e6-50a0f1867da4","order_by":0,"name":"Nathan Wood","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAm0lEQVRIiWNgGAWjYJACCYYKKIuHeC1nDEjVwthGihaD480HbxfO+yOvOyOB8cHbNmK0nDmWbD1zm4HhthsJzIZzidFidiPHTJp3mwEjUAubNC/xWuYY2AO1sP8mQUuDQSLIFmaitNiD/MJzzDh525mHzZJzzhGhRbIdGGI8NXK2244nH/zwpowILUiAsYE09aNgFIyCUTAKcAMAizc1Nuj8ByEAAAAASUVORK5CYII=","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":true,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Wood","suffix":""},{"id":341865453,"identity":"836c4a22-bb75-42c6-998b-3d3d72bc3cb9","order_by":1,"name":"Hongmin Qin","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Hongmin","middleName":"","lastName":"Qin","suffix":""},{"id":341865454,"identity":"b6c66b2f-206c-4a4e-8928-7d114152efc7","order_by":2,"name":"Wanhe Li","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Wanhe","middleName":"","lastName":"Li","suffix":""},{"id":341865455,"identity":"9aba65b9-cc56-4cd2-a4eb-d985191a2738","order_by":3,"name":"Esther Doria","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Doria","suffix":""}],"badges":[],"createdAt":"2024-07-22 21:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4784339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4784339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63066803,"identity":"1f5eaff5-67b7-4449-99e3-9a9b4b1bd1d8","added_by":"auto","created_at":"2024-08-22 17:58:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284961,"visible":true,"origin":"","legend":"\u003cp\u003eMethod to prepare alginate-methylcellulose bioink prior to addition of cells for experiments. (A) Dry baking setup for mixed alginate-methylcellulose powders. (B) Summarized protocol for preparing an aseptic bioink employing alginate and methylcellulose dissolved in a flask coating solution bearing ECM components.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4784339/v1/a3dad565b4c222633314877d.png"},{"id":63067050,"identity":"f3f05b41-d659-4e9f-8a0e-b9a3b922ab8c","added_by":"auto","created_at":"2024-08-22 18:06:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78636,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability. (A) live‐dead staining images. Viable cells are shown in blue, dead in yellow, scale bar 40 um. (B) Cell viability in four bioinks after 3, 6, and 9 days from encapsulation. Each point represents percent viability within the image taken, with three images taken per replicate. The rectangle boxes represent the interquartile range (IQR) of the experimental group’s observations, with the bisecting line representing the median observation. Vertical lines represent the sum of either the upper or lower quartile and 1.5 * IQR. N.S represents non-significance, * represents \u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e, ** represents \u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e, and *** represents \u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4784339/v1/ada0e3cc1ddf6ef3793dbf48.png"},{"id":63066801,"identity":"e4133b37-f18d-4834-9f91-6348b565231e","added_by":"auto","created_at":"2024-08-22 17:58:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82123,"visible":true,"origin":"","legend":"\u003cp\u003eCell spheroids formed in bioinks. (A) Categories used to approximate cells per aggregate. The scale bar represents 20 um. (B) Distribution of categories of spheroids across days 3, 6 and 9. Larger spheroids, such as the ones with 9-12, 13-24, and \u0026gt;24 cells became more prevalent and smaller ones become less prevalent.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4784339/v1/55742b70b0d4030e42515d76.png"},{"id":63067051,"identity":"6251d70a-8423-4902-bdf0-dbcd69dda89a","added_by":"auto","created_at":"2024-08-22 18:06:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133635,"visible":true,"origin":"","legend":"\u003cp\u003eVolume per cell in spheroid.\u003cstrong\u003e \u003c/strong\u003eNo differences in spatial confinement of cells in different bioinks was noted. Distributions of units of volume per cell for spheroids bearing 2-4 cells (A), 5-8 cells (B), 9-12 cells (C), 13-24 cells (D), and 24 or more cells (E). Groups with less than four observations were not included in statistical analysis. N.S represents non-significance, * represents p \u0026lt; 0.05, ** represents p \u0026lt; 0.01, and *** represents p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4784339/v1/25e4857e11e0b678f49b50bc.png"},{"id":65080505,"identity":"653e4e66-5803-4768-93b8-5dde92b98d2c","added_by":"auto","created_at":"2024-09-23 11:50:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1309703,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4784339/v1/e61f0758-93d2-416b-af6a-a96cfcc04ef5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Biocompatibility of 16HBE14o- Human Bronchial Epithelial Cells in Alginate-Methylcellulose Bioinks Revealed Spheroid Formation ","fulltext":[{"header":"Introduction","content":"\u003cp\u003e3D cell culture is used to address life science questions by growing cells in a 3D spatial orientation that more closely mimics their \u003cem\u003ein vivo\u003c/em\u003e environment\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. This approach allows for a more physiologically sound understanding of interactions with the cell membrane and cytoskeleton, extracellular matrix, and signal transduction. It also addresses practical problems related to tissue grafting, drug discovery, and drug delivery\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Of particular interest is the formation of cell spheroids, which are 3D cell aggregates that better mimic tissues and tumors. Unlike 2D monolayers, which are forced to adhere to a very stiff plastic or glass surface with mostly uniform exposure to metabolites and oxygen, spheroids experience metabolic gradients such as localized hypoxia, allowing for more realistic drug screenings and stress studies\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e3D bioprinting can enhance the scalability and complexity of current 3D cell culturing methods\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Extrusion-based bioprinting often employs mechanical pressure to deposit droplets or filaments consisting of a biomaterial, referred to as a bioink, onto a provided surface in a desired pattern\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Cell culturing media, in its typically used state, is unsuitable for 3D bioprinting. Therefore, it is of interest to produce materials that are viscous enough for adequate printing, while also posing minimal hazard to the cells\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDerived from brown algae, sodium alginate and its modified conjugates are popular base components for bioinks due to its low cost, biocompatibility both in vitro and in vivo, and ability to cross-link as a hydrogel when exposed to cations such as calcium\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. To enhance the physical and chemical properties of the bioink and its resulting hydrogel, and to meet the physiological needs for cells interacting with the hydrogel, additional materials, such as extracellular matrix components and cellulose derivatives are incorporated.\u003c/p\u003e \u003cp\u003eExtracellular matrix (ECM) components such as collagen and fibronectin are essential for epithelial cells, which interact with ECM via membrane integrin receptors. The absence of ECM can lead to a form of programmed death referred to as anoikis\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. To prevent this, bioinks often include ECM components or materials modified to have peptide motifs that serve as integrin ligands\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Soluble cellulose derivatives such as methylcellulose are commonly added to modify the viscosity of the bioink before cross-linking, and to modify the stiffness and compression behaviors of the hydrogel after cross-linking\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious report by Li et al. evaluated the rheological characteristics of alginate-methylcellulose mixtures at ratios of 1:3, 3:3, and 3:9 (% w/v) for extrusion bioprinting, observing greater than 95% cell viability when L929 mouse fibroblast cells were incorporated between layers of the alginate-methylcellulose bioink\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Duin et al. successfully printed a 3:9 (%) alginate-methylcellulose hydrogel laden with mouse pancreatic islets, achieving 60\u0026ndash;80% cell viability over seven days\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Ahlfield et al. demonstrated the use of an alginate-methylcellulose mixture dissolved in fresh frozen human plasma to culture mesenchymal stem cells, human dental pulp cells, and human umbilical vein endothelial cells, and human preosteoclast cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCells forming spheroids in 3D culture replicate in vivo conditions, crucial for studying disease mechanisms and drug responses with greater accuracy in biomedical research and regenerative medicine. Regarding the 3D culturing of lung cells, Celis et al. successfully formed spheroids of 16HBE14o- human bronchial epithelial cells using a hanging drop method. They demonstrated that cells in the spheroids experience oxidative stress and transcriptional shifts concerning cell-cell interactions\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. To study the effect of diesel exhaust particles on Transforming Growth Factor Beta (TGF-β) and markers of the Epithelium to Mesenchymal Transition (EMT), Baarsma et al. employed a magnetic bioprinting method in which BEAS-2B human bronchial epithelial cells were cultured with proprietary NanoShuttle\u0026trade; particles, allowing cells form spheroids by placing a magnet underneath the culture vessel\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the use of methylcellulose, Tam had employed furan-modified hyaluronic acid combined with thiolated methylcellulose to study the invasive properties of stem cell derived smooth muscle cells with loss of function in tuberous sclerosis complex 1 or 2, as a model for lymphangioleimyomatosis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Using a fibrous scaffold derived via the electrospinning of poly(ε-caprolactone) and methylcellulose, Gon\u0026ccedil;alves reported that while proliferation of 16HBE human bronchial epithelial cells are observed between 1 to 7 days, proliferation collapses between 7 and 14 days\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe study assessed the biocompatibility of methylcellulose as an alginate bioink additive for 3D culturing, and potentially3D bioprinting, of 16HBE14o- human bronchial epithelial cells. It compared cell viability and spheroid formation in bioinks containing only ionically cross-linked sodium alginate, as well as hydrogels bearing sodium alginate and methylcellulose in a 1:1 ratio (test groups shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The study found that while alginate-methylcellulose bioinks cross-linked with 300 mM calcium chloride had the lowest initial cell viability, these bioinks supported greater spheroid cell proliferation over nine days, suggesting their potential for 3D culturing. This work provides preliminary insights into using alginate-methylcellulose bioinks for 3D bioprinting, enhancing complex 3D culturing techniques.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of Bioinks\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBioink 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBioink 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBioink 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBioink 4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSodium Alginate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eECM Components\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16HBE14o-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethylcellulose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100 mM CaCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e300 mM CaCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability in Bioinks\u003c/h2\u003e \u003cp\u003eThe impact of the addition of methylcellulose and concentration of cross-linking agent on cell viability was evaluated by distinguishing living cells that fluoresce exclusively blue from dead cells that fluoresce both blue and green within each bioink (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). All four bioinks maintained cell viability over 3, 6, and 9 days, indicating that methylcellulose is not cytotoxic to 16HBE14o- cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On Day 3, all bioinks exhibited the lowest viability. By Day 6, the bioink without methylcellulose cross-linked with 300 mM CaCl\u003csub\u003e2\u003c/sub\u003e achieved the highest observed viability at 81.68%. Bioinks containing methylcellulose cross-linked with 300 mM CaCl\u003csub\u003e2\u003c/sub\u003e consistently showed the lowest viability across all days (Day 3 at 67.09%, 6 at 62.62%), and 9 at 67.76%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell Viabilities in Bioink for 3, 6, and 9 Days. All units are percentages of living cells relative to the total cells. IQR Represents the interquartile range.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e4% Methylcellulose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNo Methylcellulose\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e300 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e300 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStandard Deviation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;18.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;15.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;7.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIQR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStandard Deviation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;14.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;15.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;9.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;10.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIQR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStandard Deviation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;8.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;16.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;12.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;27.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIQR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn bioinks without methylcellulose, no statistical difference was observed in cell viability between those cross-linked with 100 mM or 300 mM CaCl2 solutions on any examined days. However, bioinks containing methylcellulose and cross-linked with 300 mM CaCl2 exhibited lower viability compared to those cross-linked with 100 mM CaCl2 on Day 3 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0096\u003c/em\u003e), but not on Day 6 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.1412\u003c/em\u003e) or Day 9 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;1.0\u003c/em\u003e). Overall, these results indicate that all four bioinks are biocompatible to 16HBE14o human bronchial epithelial cells. However, the addition of 4% methylcellulose to alginate hydrogels reduces the viability of encapsulated human bronchial epithelial cells compared to hydrogels without methylcellulose, with this reduced viability exacerbated by high concentrations of CaCl\u003csub\u003e2\u003c/sub\u003e cross-linker.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFormation of Spheroids in Bioinks\u003c/h2\u003e \u003cp\u003eOn Day 3, bioinks containing methylcellulose primarily exhibited single cells and small cell spheroids containing 2\u0026ndash;4 cells. Notable exceptions were a single 13\u0026ndash;24 cell spheroid in Bioink 1 and a single 5\u0026ndash;8 cell and 9\u0026ndash;12 cell spheroid in Bioink 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, bioinks without methylcellulose already showed a significant proportion of spheroids bearing 5\u0026ndash;8 cells by day 3. By Day 6, spheroids containing 5\u0026ndash;8 cells became the most common category across all bioinks. Spheroids with 9\u0026ndash;12, 13\u0026ndash;24, and more than 24 cells began to appear more frequently in bioinks without methylcellulose. By Day 9, spheroids with 13\u0026ndash;24 cells became the most prevalent group across all bioinks. These observations suggested that adding methylcellulose to an alginate bioink might have initially delayed the proliferation of human bronchial epithelial cells. However, this delay was overcome by Day 6. The formation of spheroids in all bioinks demonstrated their biocompatibility for the 16HBE14o- cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistributions of cell clusters in bioinks on days 3 to 9 based on cell count.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTypes of cell clusters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e4% Methylcellulose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNo Methylcellulose\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e300 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e300 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1 Cell\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u0026ndash;4 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u0026ndash;8 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u0026ndash;12 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u0026ndash;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1 Cell\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u0026ndash;4 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u0026ndash;8 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u0026ndash;12 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u0026ndash;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1 Cell\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u0026ndash;4 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u0026ndash;8 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u0026ndash;12 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u0026ndash;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;24 Cells\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSpatial Confinement of Cells in Spheroids\u003c/h2\u003e \u003cp\u003eTo analyze the spatial confinement of cells within spheroid, their volumes were estimated using the spheroid\u0026rsquo;s area under maximum intensity projection and the product of confocal Z stacks and the acquisition\u0026rsquo;s Nyquist Rate, then divided by the manually counted number of cells per spheroid. Spheroids containing smaller number of cells (2\u0026ndash;4 cells, 5\u0026ndash;8 cells, and 9\u0026ndash;12 cells) exhibited varying spatial confinement, excluding outliers, ranging approximately from 419.17 to 5728.82 \u0026micro;m\u0026sup3;cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Spheroids with 13\u0026ndash;24 cells and greater than 24 cells appeared less spatially confined, with volume per cell ranges from 1855.95 to 8649.05 and 2562.68 to 9402.33 \u0026micro;m\u0026sup3;cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe addition of methylcellulose into an alginate bioink has minimal impact on the spatial confinement of 16HBE14o- cells within a spheroid. Across 3, 6, and 9 days, all four bioinks showed consistent spatial confinement of cells within spheroids of the same cell count category (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E). An exception to this were Day 3 spheroids with 2\u0026ndash;4 cells in bioinks with methylcellulose cross-linked with 300 mM CaCl\u003csub\u003e2\u003c/sub\u003e, which were less spatially confined than those in bioinks with methylcellulose cross-linked with 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0445)\u003c/em\u003e; however, this difference were not preserved on Day 6 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;1.0\u003c/em\u003e). Additionally, the concentration of CaCl\u003csub\u003e2\u003c/sub\u003e cross-linker had negligible effect on the spatial confinement of 16HBE14o- cells, despite a previous report indicating its influence on alginate hydrogel swelling\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reports the growth of human bronchial epithelial 16HBE14o- cells into spheroids using bioinks containing alginate and methylcellulose in a 1:1 ratio. All four bioinks demonstrated suitable biocompatibility with this cell line, as accessed by cell viability and cell proliferation. Small but statistically significant effects were observed when bioinks were added with methylcellulose, which exhibited decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and a slightly impaired cell proliferation rate (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Methylcellulose serves as a viscosity modifier before cross-linking and affects stiffness and porosity after cross-linking, making it a valuable tool in 3D bioprinting for enhancing the scalability and complexity of culturing human bronchial epithelial spheroids. Future pursuits will likely explore the use of spheroids in bioinks to examine markers for epithelial to mesenchymal transition, a cancer hallmark in which epithelial cells degenerate into a multipotent and invasive state\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Additionally, pulmonary fibrosis, characterized by excessive stiffening of extracellular matrix may be simulated by increasing or decreasing bioink stiffness with different concentrations of methylcellulose and ECM components\u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. This study demonstrates the feasibility of using custom alginate and methylcellulose bioinks for 3D culture of 16HBE14o- cells, potentially mimicking complex \u003cem\u003ein vitro\u003c/em\u003e physiological conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eCell Culture\u003c/h2\u003e\n \u003cp\u003eThe 16HBE14o- (SCC150),an immortalized line of human bronchial epithelial cells, was purchased from Millipore Sigma in January 2024. Maintained in a seed-stock system, cells used in this study were used for no more than five passages since receipt from Millipore. Cells prior to mixing with bioinks were cultured in Alpha Modified Eagle Medium (\u0026alpha;-MEM, Sigma Aldrich) supplemented with Fetal Bovine Serum (10% v/v, Corning 35-010-CV Lot 27823001) and L-Glutamine (2 mM). Cells tested negative for mycoplasma contamination via confirming a lack of extranuclear Hoescht 33342 DNA fluorescence on a subsample of cells grown as a monolayer. Antibiotic-antimycotic mixture (100 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin, 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin, 0.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e amphotericin B) was not used until cells were mixed with bioinks. All cell culture was in a humidified 37\u0026deg;C incubator with 5 percent CO\u003csub\u003e2\u003c/sub\u003e supply, with media exchange performed every 48 hours.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003ePreparation of Extracellular Matrix Components\u003c/h2\u003e\n \u003cp\u003eExtracellular Matrix (ECM) components were provided by the preparation of a tissue culture flask coating solution per Millipore Sigma protocols, using Fatty Acid Free Bovine Serum Albumin (100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Cat: 126575), Advanced Biomatrix PureCol Type 1 Bovine collagen with trace Type 3 collagen (30 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Cat: 5006), and Human Plasma Fibronectin (10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Cat F2006).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003ePreparation of Alginate-Methylcellulose and Alginate Bioinks\u003c/h2\u003e\n \u003cp\u003eAlginate and methylcellulose powders were weighed separately, then mixed. The powders were then double wrapped in aluminum foil and placed in a metal tin with folded aluminum foil to separate the wrapped powders from the bottom of the tin. The tin was then covered and placed on a hot plate set at approximately 80\u0026deg;C (Fig.\u0026nbsp;1A). After two hours, the tin was allowed to cool to room temperature without intervention. After this, the powdered alginate and methylcellulose mixture was opened in a disinfected biosafety hood and exposed to the hood\u0026rsquo;s UV lamp for 15 minutes\u003csup\u003e[28]\u003c/sup\u003e (Fig.\u0026nbsp;1B).\u003c/p\u003e\n \u003cp\u003eThe flask coating solution outlined in Section 2.2.1 was added to sterile flasks with magnetic stir bars pre-cooled to 4\u0026deg;C using a serological pipette and was supplemented with antibiotic-antimycotic mixture to the concentration specified in Section 2.1. A magnetic stir plate was placed inside the biosafety hood, and the two solutions were mixed by slowly pouring in the alginate-methylcellulose mix. Once stirred until no visible particles were observed, the solutions were then stirred on the same stir plate but in a 4\u0026deg;C cold room for 36 hours. As the solutions mix, the viscosity will increase, and the qualitative stir power of the plates will need to be increased. This process was also done for a bioink with only sodium alginate (4% w/v) to serve as a control for the alginate-methylcellulose bioink.\u003c/p\u003e\n \u003cp\u003eCells grown to 80% confluence were loosened using Trypsin-EDTA (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Trypsin, 0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e EDTA) solution for 6 minutes. Cell counts were determined using a Nexcelom Cellometer Auto T4 and its proprietary two-chamber slide. 1 mL resuspended cells were added to 2 mL bioink and mixed thoroughly using a glass stir rod. The final concentration of the cell-laden bioink was 2.06x10\u003csup\u003e5\u003c/sup\u003e cell mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Cell-laden inks without methylcellulose were pipetted in 100 \u0026micro;L volumes into wells of a six well plate, each containing a glass coverslip, while cell-laden inks with methylcellulose were transferred onto glass coverslips in wells using a metal scoopula. All inks were cross-linked in 500 \u0026micro;L of either 100 mM or 300 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution in water. Cross-linking was done in a delayed sequence such that no individual bioink was bathed for more than 5 minutes. All experimental groups were done in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eData and Image Analysis\u003c/h2\u003e\n \u003cp\u003eImages were processed using a Fiji is Just ImageJ (FIJI) version 2.15.1. Any observations of drift along the Z-stack were corrected by the Correct_3d_Drift.py script provided as an intrinsic FIJI plugin. Linear adjustments to brightness and contrast were performed with GIMP 2.10.34.\u003c/p\u003e\n \u003cp\u003eStatistical analysis was conducted using LibreOffice Calc 7.4.7.2 and Rstudio version 2023.12.1\u0026thinsp;+\u0026thinsp;402 with R version 4.2.2 Patch r83330. Levene\u0026rsquo;s Test is provided as part of the R package car 3.1-2. Dunn\u0026rsquo;s Post-Hoc Test is provided as part of R package dunn.test 1.3.2. Visualization was accomplished using ggplot2 3.5.1 and ggsci 3.1.0.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eEvaluating Cell Viability Using Epifluorescent Microscopy\u003c/h2\u003e\n \u003cp\u003eCell-laden bioinks, cross-linked and incubating in culture media, were treated with Invitrogen ReadyProbes Blue/Green Viability Imaging Kit (R37609, Thermo Fisher), containing Hoescht 33342 to stain all cells, and NucGreen Dead, which only permeates the compromised membranes of dead or dying cells. Two drops of each solution were added to each well. Living cells should exclusively fluoresce blue, whereas dead and dying cells should fluoresce green and blue (Fig.\u0026nbsp;2A). Cells were then incubated for 30 minutes. Cells were imaged using an Echo Revolution microscope in its inverted configuration, with an incubator chamber mated to the microscope set to 37\u0026deg;C, and given a 5 percent CO\u003csub\u003e2\u003c/sub\u003e, 20 percent O\u003csub\u003e2\u003c/sub\u003e mixture with 150 cc flow rate. Images were taken using a dry infinity corrected Olympus PlanLUC FLN 10X/0.3 using Chroma DAPI and FITC filters and a monochrome sCMOS detector and acquisition software (version 2.0.0.0) intrinsic to the microscope.\u003c/p\u003e\n \u003cp\u003eFor analysis of cell viability, the Kruskal-Wallis statistical test followed Dunn\u0026rsquo;s post-hoc test with comparisons adjusted using Bonferroni correction. P values below 0.05 were considered significant. The Kruskal-Wallis test was chosen as the distribution of residuals for test groups did not follow a normal distribution both graphically and per the Shapiro-Wilk\u0026rsquo;s Test (lowest \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.000395\u003c/em\u003e ), while maintaining homogeny of variance using Levene\u0026rsquo;s Test (lowest \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0829\u003c/em\u003e). On plots, statistical equivalence is abbreviated as Not Significant (N.S), whereas significance is abbreviated as * (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), ** (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), or *** (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eEstimating Number of Cells in Spheroids\u003c/h2\u003e\n \u003cp\u003eSamples were washed three times with 1X Phosphate Buffered Saline, then fixed with paraformaldehyde in 1X PBS solution (4% w/v) for 2 hours. Samples were then washed three times again with 1X PBS then treated with SYTOX Green nucleic acid stain (1:2000 dilution, Thermo Fisher) to stain cell nuclei for 30 minutes in the dark. Samples transferred to a microscope slide and treated with Invitrogen SlowFade glycerol mountant. Coverslips were placed on top of the bioink samples prior to imaging with a Zeiss Examiner.Z1 with LSM900 Laser Scanning Confocal microscope, in frame scanning mode with 488 nm excitation laser and a Zeiss Plan Apochromat 20X/0.8 dry objective. When examining the number of cells within an spheroid, the approximate number of cells was counted manually and then each respective cell count was organized into categories of 1, 2\u0026ndash;4, 5\u0026ndash;8, 9\u0026ndash;12, 13\u0026ndash;24, and greater than 24 cells, visualized in Fig.\u0026nbsp;3A and quantified in Table\u0026nbsp;3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eEstimating Volume of Spheroids\u003c/h2\u003e\n \u003cp\u003eTo examine if the addition of methylcellulose or different concentrations of cross-linking agents impacts how spatially confined cells are within spheroids, the approximate volume of observed spheroids was divided by the number of cells found in the particular spheroid. Axial height of a spheroid was approximated using Z stacks. Lower and upper Z sections were selected from when a given spheroid becomes totally out of focus, with the difference begin multiplied with the acquisition\u0026rsquo;s Nyquist Rate (0.53 \u0026micro;m). Using the area determined from the maximum intensity projection of the Z stack and the axial height, the volume of a spheroid was calculated as follows.\u003c/p\u003e\n \u003cdiv id=\"Equ1\"\u003e\n \u003cdiv id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:V=\\frac{4}{3}\\pi\\:{r}^{2}c$$\u003c/div\u003e\n \u003cdiv\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eWhere:\u003c/p\u003e\n \u003cp\u003e- \\(\\:\\pi\\:{r}^{2}\\) represents the area determined from a maximum intensity projection\u003c/p\u003e\n \u003cp\u003e- \\(\\:c\\) is the axial radius, or half of the axial height, as calculated from upper and lower Z stacks\u003c/p\u003e\n \u003cp\u003eThe Kruskal Wallis Test with Dunn\u0026rsquo;s post-hoc test with Bonferroni correction was used to compare units volume per cell. Groups in which there were less than four observations were discarded from future analysis. This decision was made as the distribution data (spheroidal volume per cell) appears to not be normal per Shapiro-Wilk test (lowest \u003cem\u003ep\u0026thinsp;=\u0026thinsp;1.026x10\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;5\u003c/em\u003e\u003c/sup\u003e), while still maintaining homogeny of variance per Levene\u0026rsquo;s Test (lowest \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.1011\u003c/em\u003e, groups below 0.05 had sample sizes less than four and were discarded). Statistical significance on plots is designated in the same manner for cell viability in Section 2.3.1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eData Release Statement\u003c/h2\u003e\n \u003cp\u003eData used in this report, including image data, statistical reports, and tabulated data can be provided upon reasonable request by the corresponding author.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to report.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.W conceived the experiments, prepared bioinks, lead image acquisition, and analyzed data. E.D. assisted with confocal microscopy. W.L. provided the Zeiss Examiner.Z1 and LSM 900 Unit. Z.P. is the co-principal investigator, provided research advisory, and reviewed the manuscript. H.Q. is the principal investigator, provided research advisory, and reviewed the manuscript. All authors have received a copy of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was funded by the U.S. Air Force Office of Scientific Research (AFOSR), grant number FA9550-23-1-0599, and FA9550-23-1-0156. W.L is a CPRIT Scholar in Cancer Research (RR220021)\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData used in this report, including image data, statistical reports, and tabulated data can be provided upon reasonable request by the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHaycock, J. W. 3D cell culture: a review of current approaches and techniques. Methods Mol Biol 695, 1\u0026ndash;15 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNunes, A. S., Barros, A. S., Costa, E. C., Moreira, A. F. \u0026amp; Correia, I. 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APL Bioeng 5, 046102 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHodder, E. \u003cem\u003eet al.\u003c/em\u003e Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes. Journal of Materials Science: Materials in Medicine 30, (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"3D, spheroid, epithelium, bioink, alginate, methylcellulose","lastPublishedDoi":"10.21203/rs.3.rs-4784339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4784339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe biocompatibility of 16HBE14o- human bronchial epithelial cells in ionically cross-linked alginate-methylcellulose bioinks was assessed. This was accomplished by encapsulating 16HBE14o- cells in either a sodium alginate bioink or a bioink with sodium alginate and added methylcellulose in a 1:1 ratio. To differentiate the effects of methylcellulose from those of cross-linking on cell viability, two concentrations of calcium chloride cross-linker were used for both alginate only and alginate-methylcellulose bioinks. Using fluorescence microscopy, it was observed that bioinks with methylcellulose showed a small but significant reduced cell viability and a decreased presence of cell spheroids compared to their methylcellulose free alginate counterparts. However, alginate-methylcellulose bioinks still supported cell proliferation and appeared to be biocompatible. Additionally, the concentration of cross-linker seemed to impact cell viability. This study has implications for the use of methylcellulose as a viscosity tuner for both general 3D 16HBE14o- human epithelial cell culture and 3D bioprinting. The presence of spheroids suggests that alginate-methylcellulose bioinks could be useful in generating 3D 16HBE14o- human epithelial cell culture to address questions in cell biology, including signal transduction, metabolic activity, and cancer hallmarks.\u003c/p\u003e","manuscriptTitle":"Assessment of Biocompatibility of 16HBE14o- Human Bronchial Epithelial Cells in Alginate-Methylcellulose Bioinks Revealed Spheroid Formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 17:58:49","doi":"10.21203/rs.3.rs-4784339/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7abffdd8-408e-4386-af80-39229cd541b2","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36215452,"name":"Biological sciences/Biological techniques/Cytological techniques/Cell culture"},{"id":36215453,"name":"Biological sciences/Biological techniques/Biological models/Respiratory system models"}],"tags":[],"updatedAt":"2024-09-23T11:42:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 17:58:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4784339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4784339","identity":"rs-4784339","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}