Engineering Biocompatible Polylactic Acid (PLA) Microcarriers for Enhanced 3D Cell Culture

doi:10.21203/rs.3.rs-7708554/v1

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Engineering Biocompatible Polylactic Acid (PLA) Microcarriers for Enhanced 3D Cell Culture

https://doi.org/10.21203/rs.3.rs-7708554/v1

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This study analyses the performance of polylactic acid (PLA)-collagen microcarriers engineered under laboratory-scale conditions with scalable potential for three-dimensional (3D) cell culture applications. The microcarriers were evaluated for their ability to support cell adhesion, viability, proliferation, and conglomerate formation using Caco-2 cells. Results show that PLA-collagen microcarriers significantly improve cell density, with notable proliferation by day 7. Scanning Electron Microscopy (SEM) revealed a rough, porous surface topology that fosters cellular aggregation and conglomerate formation, essential for replicating complex tissue architectures. Multigenerational monitoring using epifluorescence assays demonstrated high nuclear density and sustained cell viability, further supporting the microcarriers' ability to maintain dense and viable cellular environments. Additionally, Energy Dispersive X-ray Spectroscopy (EDS) confirmed effective cell-microcarrier interactions, validating the bioactivity of the PLA-collagen matrix. These findings underscore the potential of PLA-collagen microcarriers as a versatile and scalable tool for 3D cell culture, with significant applications in bioengineering tumor models, organoid formation, and drug testing. Technology offers a promising approach for advancing preclinical research and regenerative medicine.

3D cell culture

Polylactic acid

Microcarriers

Bioengineering

Conglomerate formation

organoids

Epifluorescence

SEM (Scanning Electron Microscopy)

Since the introduction of conventional eukaryotic cell culture nearly five decades ago, the most commonly used support for promoting cell growth has been manufactured from polystyrene or glass in a flat, two-dimensional (2D) format (Freshney, 2015). Numerous studies, ranging from the examination of antineoplastic agents to developmental biology, have relied on the 2D format for the adhesion and cultivation of animal cells. However, these studies have been criticized because they assume that animal physiology can be sufficiently replicated by a cellular monolayer (Haycock, 2011).

Three-Dimensional (3D) culture systems

It is now widely recognized that culturing eukaryotic cells on 2D substrates fail to replicate the complex architecture of the native extracellular matrix (ECM) found in tissues (Pampaloni, Reynaud, & Stelzer , 2007). Consequently, marked variability has been documented in a range of complex biological responses, such as receptor expression, cell migration and apoptosis, when compared to those in the original organ or tissue (Haycock, 2011).

Three-dimensional (3D) culture matrices were developed to address the architectural limitations of 2D cultures. These porous matrices support growth, organization and even cellular differentiation within their structure (Lee, Cuddigy , & Kotov, 2008). A distinctive feature of 3D models is the intricate interplay between cells and their local environment, including both cell-cell and cell-matrix interactions, which collectively aim to emulate tissue organization (Kapałczyńska, et al., 2018; Lee, Cuddigy , & Kotov, 2008). Within this system, cells respond to various stimuli in a manner resembling in vivo conditions, particularly in terms of topology, gene expression and metabolism (Cawkill & Eaglestone, 2007). Furthermore, 3D cultures systems help maintain cell morphology and polarity and offer cells the opportunity to regain native phenotypic characteristics, even if they were previously cultured on 2D surfaces (Yamada & Cukierman, 2007).

Microcarriers and Three-dimensional cell cultures

Microcarriers are small particles, typically 50-400 µm in diameter, used as platforms for culturing cells in suspension. They have found broad utility in cell manufacturing, drug delivery and advanced cell culture applications (Farid & Jenkins, 2018). Their customizable design and versatility have made them indispensable in biomedical fields such as tissue engineering, 3D bioprinting and disease modelling (Handral, Wyrobnik, & Lam, 2023).

When incorporated into 3D culture system, microcarriers function as scaffolds that promote cell attachment, proliferation and, in some cases, differentiation within a volumetric environment more akin to physiological conditions than standard 2D cultures (Derakhti, Safiabadi-Tali, Amoabediny, & Sheikhpour, 2019). By supporting the formation of spheroids or multilayered cell clusters that resemble native tissue architecture, microcarriers are useful for applications that require high cell yields or precise spatial organization, such as studies on stem cell maturation and tumor biology (Xiao, Ehsanipour, Sohrabi, & Seidlits, 2018).

A range of materials can be used to fabricate microcarriers, including metals, ceramics, glasses, polymers and biologically derived substances, as well as blends of these materials (Muschler, Nakamoto, & Griffith , 2004). Polymers, especially biocompatible and biodegradable varieties such as polylactic acid (PLA) and polyglycolic acid (PGA), are widely employed because their chemical and structural attributes can be finely tuned (Lee, Cuddigy , & Kotov, 2008). This adaptability permits precise control over mechanical strength, degradation rate and surface properties. In addition, various manufacturing techniques allow for microcarrier fabrication on both microscopic and nanoscopic scales, enhancing their suitability for numerous biomedical contexts (Urwyler & Schift, 2016). PLA, in particular, is a prominent choice due to its FDA approval, renewable resourcing, and sustainability for medical applications (Kohn, Welsh, & Knight, 2007). Common production methods for PLA microcarriers, such as electrospinning and solvent emulsification, facilitate tight control over physical characteristics to meet specific cellular requirements (Costa, Pereira, Silva, Jesus, & Souza Jr, 2018).

For clinical applications, biodegradable materials must be chosen so that any byproducts of microcarrier degradation are non-toxic and do not provoke immune responses or fibrous encapsulation (Haycock, 2011). Furthermore, microcarriers destinated for therapeutic use must fulfil stringent pharmacopeial standards for sterility and safety (Costa, Pereira, Silva, Jesus, & Souza Jr, 2018).

Collagen surface coating

Collagen, a primary component of ECM, is vital to many cellular processes such as attachment, migration, proliferation and differentiation, owing to its biocompatibility and biodegradability (Zhou, Liu, Zhang, Gu, & Peng, 2023). Its structural and biochemical properties make it ideal material for bioengineering applications, particularly for upgrading the performance of scaffolds and drug delivery systems. In tissue engineering, incorporating collagen into PLA scaffolds has been shown to markedly improve cellular adhesion, spreading and proliferation (Santoro, Shah, Walker, & Mikos, 2016). Notably, collagen-coated PLA scaffolds increase cell viability, DNA content and metabolic activity in human dermal fibroblasts, compared with unmodified PLA (Cui, Liu, Guo, Su, & Ma, 2015).

Collagen coatings on PLA scaffolds can also enhance osteogenic differentiation in stem cells, making them suitable for bone tissue engineering (Stratton, Shelke, Hoshino, Rudraiah, & Kumbar, 2016; Xiao Y. , et al., 2007). The low immunogenicity and high biocompatibility of collagen allow it to be recognized by the body as a natural component, thus diminishing adverse immune responses and broadening its use in therapeutic contexts (Sehgal & Srinivasan, 2009). These attributes have further enabled the development of collagen-based particles for drug delivery, facilitating controlled release for localized therapy.

Moreover, modifications to the surface of microcarriers expand the utility of collagen. Surface modifications can improve drug-loading capacity and release kinetics, enabling targeted delivery to specific cells or tissues (Handral, Wyrobnik, & Lam, 2023). Additionally, coatings with ECM protein —such as collagen, fibronectin, vitronectin, or their peptide motifs—have been shown to enhance cytoskeletal organization, modifying cell morphology, and influence cellular behavior through activation of intercellular signaling pathways and changes in gene expression (Bello, Kim, Kim, Park, & Lee, 2020; Nath, Harper, & Rancourt, 2020; Yin & Cao, 2021). Taken together, these properties underscore collagen’s versatility in scaffold engineering and drug delivery applications.

Three-dimensional Cancer Models

Employing 3D cell culture systems constitutes a significant advancement in biomedical research by providing a multifaceted means to study critical aspects of comparative biology. A key advantage of 3D culture systems is their capacity to closely mirror the intricate interplay between cells and the ECM, thus approximating the native architecture of tumor masses (Kapałczyńska, et al., 2018). This fidelity is crucial, given the established link between ECM composition and multiple aspects of cancer progression and tumor phenotypes. Through 3D systems, researchers can more accurately evaluate drug sensitivity and resistance, reducing the risk of underestimating or overestimating drug efficacy and dosage (Griffith & Swartz, 2006; Cawkill & Eaglestone, 2007).

Furthermore, the formation of spheroids within 3D cultures offers a robust platform to investigate processes such as cell migration, ECM protein invasion and the coordinated interaction between tissue invasion and angiogenesis (Kapałczyńska, et al., 2018). This in turn shed light on the mechanism of invasion, metastasis, tumor-microenvironment interactions and therapeutic resistance (Arneth, 2019).

Equally important, 3D culture systems facilitate the generation of synthetic cancer tissues that reproduce critical tumor features, including the expression of specific biomarkers (Kapałczyńska, et al., 2018). Such models offer unparalleled opportunities to investigate tumor biology in a physiologically relevant context, supporting detailed studies of tumor behavior and response to treatments.

Within the realm of colorectal cancer (CRC), a major global health concern, the development of precise models is imperative for advancing treatment efficacy (Smietana, Siatkowski, & Moller, 2016; Bhattacharjee, 2012). The complex relationship between the tumor microenvironment and the hierarchy of cancer cells remains an area of active investigation (Kawai, et al., 2020).

Scanning Electron Microscopy and Energy-dispersive X-Ray Spectroscopy

Scanning Electron Microscopy (SEM) is employed as a complementary technique to characterize scaffolds and proliferation on the extracellular matrix. SEM is used to examine and characterize a material’s nanostructure and morphology (Karak, 2019). This method focuses an electron beam onto the sample with voltages of up to 30 kV, the interactions between the beam and the sample generate signals that are detected, recorded and converted into grayscale images (Rodríguez-Herrero, Lopez-Camas, & Ullah, 2023) (Xiao K. , Xu, Cao, Xu, & Li, 2022). SEM is often used to determine particle size and pore dimensions in collagen scaffolds (Chakraborty, Asthana, Singh, Adhikari, & Hasan, 2023).

Because polymers and other materials may exhibit low conductivity, a conductivity coating -typically gold or copper- is commonly applied prior to SEM analysis (Heu, Shahbazmohamadi, & Yorston, 2019). In contrast, biological samples require chemical fixation to preserve their architecture and bioactive components by imparting mechanical rigidity and preventing decomposition (Thavarajah, Mudimbaimannar, Elizabeth, Rao, & Ranganathan, 2012) (Al Shehadat, et al., 2018). Formaldehyde and glutaraldehyde are frequently used as fixatives, followed by organic solvents for dehydration to minimize artifacts in the resulting images (Thavarajah, Mudimbaimannar, Elizabeth, Rao, & Ranganathan, 2012).

An elemental analysis know as Energy-Dispersive X-Ray Spectroscopy (EDS) is often coupled with SEM. EDS reveals the elemental composition of a sample and provides a map of each element present in the spatial distribution (mapping) of each element by scanning an electron beam across the sample’s surface while X-Rays emitted from the sample are detected and collected by an energy dispersive spectrometer (Samal, et al., 2024) (Gupta, Semwal, & Pathak, 2020).

When the sample is excited by the incident electron beam, it releases energy in the form of X-Rays and it is characteristic for each atomic structure of an element, which is detected at approximately 2 mm deep from the sample’s surface, providing a spatial distribution bidimensional mapping of the analyzed surface based on the energy signals received by the spectrometer (Kumar, Balaji, & Mandlimath, 2023) (Shojaei, Soltani, & Derakhshani, 2022).

Study overview

This proof-of-concept study provides a comprehensive assessment of polylactic acid (PLA)-collagen microcarriers as an alternative to commercially available microcarriers for 3D cell culture. PLA-collagen microcarriers, produced in laboratory-scale bioreactors with the potential for scale-up, were evaluated for their capacity to promote cell adhesion, viability, proliferation and cellular conglomerate formation. A combination of cell viability and proliferation assays, multigenerational monitoring via epifluorescence microscopy, and characterization by SEM and EDS was used to evaluate morphological properties, collagen coating and cell-microcarrier interactions. The findings indicate that PLA-collagen microcarriers support high cell density, robust adhesion and enhanced aggregation, positioning them as a promising alternative to existing commercial microcarriers for applications requiring a resilient 3D culture environment.

PLA microcarriers production by emulsion process

PLA microcarriers (MCs) were synthesized using an emulsion-solvent evaporation method as reported by Hong et al. (2005) (Hong, Gao, Xie, Gong, & Shen, 2005). Briefly, 3 g of PLA were dissolved in 60 mL of methylene chloride (CH₂Cl₂, Washington, USA) to create a dispersed phase. This solution was then introduced into 300 mL of deionized water containing 0.5% (w/v) polyvinyl alcohol (PVA, Fagalab, USA) under continuous agitation at 2 rfc for 24 hours at 25°C. This step facilitated evaporation of the organic solvent. The oil-to-water ratio was maintained at 1:5.

The resulting MCs were collected on filtration membranes with a pore size of 100 µm (Corning, USA) and rinsed thoroughly with sterile deionized water to remove any residual impurities. The emulsification process provided an 80% recovery of MCs. Subsequently, microcarriers were sterilized using a standard autoclave cycle (121°C, 15 psi, 15 minutes) (Boursier, et al., 2018), separated and stored under sterile conditions for further experiments.

Collagen surface coating of PLA microcarriers

Collagen cross-linking on PLA microcarriers was achieved via a dehydrothermal (DHT) treatment protocol (Weadock, Miller, Bellincampi, Zawadsky, & Dunn, 1995). First, a collagen solution was prepared using 50 mM Tris, 50 mM NaCl, 0.36 mM CaCl₂, 5 mM EDTA, 20 mg of bovine collagen type I (C9879-5G, Sigma-Aldrich, Massachusetts, USA) and deionized water (Merck, Darmstadt, GE). This solution was stirred at 35°C for 24 hours and its pH was adjusted to 7.4. Following dissolution, the solution was filtered through a 0.22 µm membrane to ensure sterility.

Subsequently, PLA microcarriers were immersed in the collagen solution for 15 minutes under aseptic conditions (Class II Type A2, ESCO, Singapore). The microcarriers were then dried in an anaerobic chamber (855-AC, Lansing, Michigan, USA) at 70°C and 17 inHg for 96 hours to facilitate dehydrothermal cross-linking. This process produced structurally robust, biocompatible microcarriers suitable for biomedical applications (Chen, et al., 2020).

Sterility tests on PLA microcarriers

To confirm sterility, microcarriers were incubated in LB agar (Sigma-Aldrich, Massachusetts, USA) and MRS agar (Thermo Fisher Scientific, Massachusetts, USA) at 35°C for 144 hours to check for microbial growth. A second test was performed under standard cell culture conditions (DMEM at 37°C, 5% CO2 and 90–95% relative humidity). All tests were conducted in triplicate. Negative controls (sterile media without microcarriers) ensured medium sterility, whereas positive controls (non-sterile PLA microcarriers) verified the efficacy of the sterility detection setup. According to USP 71 guidelines, bacterial growth should appear within 96 hours and fungal growth within 144 hours if contamination is present. No microbial growth was observed after 5 days, confirming successful sterilization (Convention, 2018; Europe, 2018).

Characterization of PLA-Collagen Microcarriers

Microcarriers morphology and compositions were assessed via SEM (JSM-6010LA, JEOL, Japan) operated at 15 kV. Prior to SEM, samples were sputter-coated with a 10 nm-thick gold layer (EM ACE200, LEICA, Germany) to enhance secondary electron emission and mitigate charging effects in non-conductive samples (Keles, Naylor, Clegg, & Sammon, 2015). The microcarriers were mounted on carbon tape, to ensure a stable, conductive substrate for high-quality imaging. SEM images were used to evaluate morphological parameters (particle sizes, pore diameter) and a control group of Corning Collagen Coated microcarriers (Corning, Massachusetts, USA) served as a reference. Representative images (n = 120) were analyzed in Image J (Version 1.50h, NIH, U.S); mean values ± standard error for particle size and pore diameter are presented in Table 1.

Caco-2 cell culture

Human adenocarcinoma colorectal cells (Caco-2; ATCC, Virginia, USA) were cultured according to Lea (2015) (Lea, 2015). Cells were maintained in DMEM (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Wisent, Quebec, CA), 1% L-glutamine (100X, Gibco), 1% non-essential amino acids (10 mM, Gibco), and 1% streptomycin/penicillin (10,000 µg/mL, Gibco). Cultures were held at 37°C in 5% CO2 and 90–95% relative humidity. When cells reached 80–90% confluency, they were harvested using 0.25% trypsin-EDTA (Gibco) for 3–5 minutes at 37°C. The cell suspension was centrifuged at 259 rfc for 5 minutes at 17°C and the pellet was resuspended in growth medium. Cell counts and viability were determined by trypan blue dye exclusion (Piccinini, Tesei, Arienti, & Bevilacqua,, 2017).

Analysis of cell viability on PLA-Collagen microcarriers

A 10-day cell proliferation kinetics study was conducted in 96-well ultralow-attachment plates (NEST, Jiangsu, China) to minimize nonspecific adhesion (Hu, Li, Huang, Wang, & Han, 2021; Chen, Chen, Choo, Reuveny, & Oh, 2011). Caco-2 cells were subcultured and seeded alongside the microcarriers in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids and 1% streptomycin/penicillin. The seeding density was optimized at 22,500 cells per well with a microcarrier concentration of 14 g/L; Corning Collagen Coated microcarriers served as the control group. Cell viability and proliferation were measured by trypan blue exclusion, assessing both microcarrier-adherent cells and overall cluster viability.

Dual-Staining approach to study cell distribution and proliferation

A standard CMFDA (5-chloromethylfluorescein diacetate) labelling protocol (CellTracker®, Thermo Fisher Scientific, Massachusetts, USA) was employed (Zhou, et al., 2016) (Bergenheim, et al., 2019). Upon internalization, the dye is converted into a stable fluorescent compound that is retained within cells through multiple generations, making it ideal for tracking cell proliferation. Fluorescence remains confined to the labelled cells and their progeny, without transferring to adjacent cells in the population. The probes retain fluorescence for at least 72 hours, under physiological conditions (Zhou, et al., 2016). Additionally, nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) to enhance the visualization of cellular structures. Endpoint dual-staining was performed following the 10-day proliferation kinetics experiment to assess cell distribution, viability, and cluster formation on the microcarriers.

Imaging was performed using an inverted optical microscope (Zeiss AxioVert) equipped with FITC (492 nm) and DAPI (358 nm) filters optimized for fluorescence microscopy. This setup allowed precise visualization of cellular structures and interactions within the microcarrier culture system.

Cell-microcarrier biointerface visualization

To evaluate cell cluster formation and the cell-microcarrier biointerface, a 10-day growth kinetics study was carried out. Caco-2 cells were seeded at 22,500 cells per well, with microcarriers at 14 g/L, in 96-well ultralow-attachment plates (NEST). Corning Collagen Coated microcarriers served as controls. Cultures were maintained under the conditions described above. On days 3, 7, 10, 12 and 14, clusters were fixed with 4% paraformaldehyde (Sigma-Aldrich, Massachusetts, USA) for SEM analysis (JSM-6010LA, JEOL, Japan) at 15 kV. Prior to SEM imaging, samples were sputter-coated with a 10 nm-thick gold layer (EM ACE200, LEICA, Germany). Comparison was made between these SEM micrographs and the cell proliferation and multigenerational data to provide a comprehensive overview of cell-microcarrier interactions.

Caco-2 cells were identified using energy-dispersive X-ray Spectroscopy (EDS) based on sodium detection, leveraging the know role of NA+,K+-ATPase in Caco-2 cells (Vieira-Coelho & Soares-da-Silva, 2001) (Hurni, et al., 1993). In parallel, microcarriers were identified by mapping for carbon and oxygen, which are predominant in PLA and Collagen (Kudzin & Mrozinska, 2020) (Leon-Mancilla, Araiza-Téllez, Flores-Flores, & Pia-Barba, 2015).

Statistical analysis

All results are presented as mean ± standard error. Statistical analyses were performed using GB-STAT Version 7.0 program (Dynamic Microsystems, Silver Spring, MD). Probability values were calculated as appropriate and p-values below conventional thresholds (e.g., 0.05) were considered statistically significant. Data analysis was performed using Welch’s test (n = 120, for each microcarrier group), additionally a two-way repeated measures ANOVA was performed for the Kinetics of cell adhesion experiment.

PLA-Collagen microcarrier analysis by Scanning Electron Microscopy

SEM was employed to compare the morphological features of PLA-Collagen microcarriers (MCs) with commercial Corning MCs, focusing on particles and pore size distribution. SEM images of PLA-Collagen MCs revealed distinct structural characteristics, including smaller particle sizes and narrower pore regions relative to Corning MCs, findings that may influence cell adhesion and proliferation.

As show in Figure 2A – C, PLA-Collagen MCs exhibited a spherical shape with a rough, porous surface. Higher-magnification images unveiled an interconnected network of pores and crevices, indicative of a heterogeneous pore-size distribution. This topography is expected to enhance cell attachment, proliferation and differentiation, suggesting potential applicability in tissue engineering. Additionally, PLA-Collagen MCs displayed a wider range of particular size, indicating a greater degree of structural heterogeneity.

In contrast, Corning MCs (Figure 2D – F) had a smooth, non-porous surface and a uniform spherical shape. The lack of surface irregularities and porosity may provide fewer binding sites for cells, potentially reducing their capacity to support cell attachment and growth.

These results highlight notable surface-property differences between the two microcarriers types. The rough, porous surface of PLA-Collagen MCs is likely to promote favorable cell-material interactions, whereas the smooth surface of the Corning MCs may limit such interactions, especially in applications where robust cell adhesion and tissue formation are required.

Particle size measurement revealed that PLA-Collagen MCs were smaller on average than Corning MCs. In particular, the diameter frequency distribution (Figure 3) showed that most PLA-Collagen MCs ranged from 30 to 50 μm, with an average diameter of 45.6 μm ± 2.3 μm, whereas Corning MCs ranged from 70 μm to over 210 μm and an average diameter of 171.1 μm ± 2.1 μm. Table 1 provides descriptive statistics for both microcarrier types.

Table 1. Descriptive statistics analysis for PLA-Collagen and Corning microcarriers following particle analysis with Scanning Electron Microscopy.

Microcarrier type	n	Avg. Diameter (μm)	SD (μm)	SE Mean (μm)
PLA-Collagen	120	45.6	25.5	2.3
Corning	120	171.1	23.0	2.1

An analysis of average particle diameter (Figure 4) confirms that PLA-Collagen MCs are significantly smaller than Corning MCs. Welch’s t-test applied due to unequal variances, identified a statistically significant difference in mean particle diameters between the two groups (p < 0.0001, t = 40.02, df = 235.5). The 95% confidence interval for the difference in mean diameters (119.2 and 131.5 μm) underscores the large magnitude of this discrepancy and an eta-squared value of 0.8718 indicated a strong effect size. These data suggest that the differences in particle size could substantially influence experimental outcomes, depending on which microcarrier is selected.

Kinetics of cell adhesion and proliferation on microcarriers

A 10-day kinetics proliferation study (Figure 5) was conducted to evaluate cell growth and conglomerate formation on PLA-Collagen MCs in comparation to commercial MCs (Corning). Both microcarrier types were seeded at the same initial cell density (22,500 cells/well, 14 g/L MCs) to ensure standardized starting conditions.

A two-way repeated measures ANOVA (α = 0.05) was performed for this experiment, revealing significant main effects for both the microcarrier type and the day of kinetics, as well as a significant interaction between these factors. Microcarrier type accounted for the largest variation (43.06%, p = 0.0167), with PLA-Collagen microcarriers supporting significantly higher adhesion (19,013 ± 2,386) compared to commercial microcarriers (9,563 ± 2,386). Time (row factor) contributed 25.30% of the variation (p = 0.0006), reflecting changes in adhesion dynamics over days 1, 4, 7, and 10, while the interaction (15.90%, p = 0.0004) highlighted the distinct adhesion and proliferation profiles of the two microcarrier types over the study period.

By Day 1, both groups showed a reduction in cell numbers, reflecting incomplete cell adhesion during the first 24 hours. However, PLA-Collagen MCs retained 50% of the initial cell density (11,250 cells/well), compared to only 39% on Corning MCs (8,775 cells/well). This higher retention suggests that PLA-Collagen MCs provide a superior surface for initial cell attachment.

By Day 7, the Welch’s test (p < 0.05; Figure 5) reveals a highly significant difference between the two microcarrier types, with PLA-Collagen microcarriers exhibiting a much higher mean cell count (28,350 cells/well) compared to the commercial microcarriers (9,900 cells/well). The effect size (R² = 0.9344) underscores the substantial impact of the microcarrier type on cell adhesion and proliferation. Variances between the groups were not significantly different, supporting the validity of the test. These results indicate that PLA-Collagen MCs create a more favorable microenvironment for cell adhesion and proliferation, particularly during the mid-phase of the kinetics experiment.

On Day 10, cell density declined in both groups, likely because of nutrient depletion and waste product accumulation in the culture, as well as space limitations.

Dual-Staining approach to study cell distribution and proliferation

Epifluorescence microscopy was used on Day 10 to assess Caco-2 cell adhesion, proliferation and conglomerate formation on PLA-Collagen versus Corning MCs. DAPI staining (blue) labelled cell nuclei and CMFDA (green) indicates viable cells. This dual-staining approach allowed visualization of both cell viability and nuclear distribution across microcarrier surfaces. Moreover, the metabolization and conversion of CMFDA confirmed ongoing cell divisions (Figure 6).

On the PLA-Collagen MCs, DAPI staining (Figure 6A) revealed a high nuclear density of cell nuclei, consistent with substantial cell attachment. CMFDA staining (Figure 6B) confirmed cells viability showing bright green, fluorescent cells. Bright-field images (Figure 6C) indicated the formation of large cellular aggregates that densely covered the microcarrier surface. This clustering points to a favorable environment for Caco-2 cells, enabling high cell density and confluency. The robust cell adhesion and proliferation observed in these images underscore the utility of PLA-Collagen MCs for applications requiring vigorous cell growth.

In contrast, Corning MCs supported fewer attached cells (Figures 6D and 6E) with sparse cell densities and only limited patchy adherence. Fewer nuclei were evident in DAPI-staining images and CMFDA fluorescence indicated lower viability and minimal replication. Bright-field images (Figure 6F) corroborated these observations, revealing little to no clustering. Numerous cells appeared to be detached and nonviable on the culture plate, reflecting limited potential for robust cell adhesion and proliferation on these microcarriers in this setup.

Overall, the epifluorescence and bright-field imaging demonstrate that PLA-Collagen MCs provide superior conditions for cell adhesion, proliferation and clustering compared to Corning MCs.

Cell-microcarrier biointerface visualization

SEM analysis was performed on both microcarrier types to observe the ECM features and highlight critical physical attributes that mat govern cell-material interactions. As discussed previously, PLA-Collagen MCs (Figure 2A - B) display a slightly irregular, highly porous surface with smaller particle size, whereas Corning MCs have a smooth surface, a defined spherical shape and larger overall diameter (Figure 2C - D).

SEM imaging of PLA-Collagen MC cultures was conducted on Days 3, 7, 10, 12 and 14 (Figure 8) where cells were seeded in a 6-well plate at an initial density of 135,000 cells/well and 14 g/L of PLA-Collagen MCs.

Day 4 (Figure 8A): single cell adhesion on the ECM was visible, marking early-stage cell proliferation on the microcarriers.

Day 7 (Figure 8B): a multicellular cluster began forming, linking multiple MCs in a spherical arrangement.

Day 10 (Figure 8C): clusters increased in size and number compared to Day 7.

Day 12 (Figure 8D): further increases in cluster density and overall size were evident on the MCs surfaces.

Day 14 (Figure 8E, 8F): the largest and densest cell cluster were observed, including morphological changes suggestive of early enterocyte-like features.

Under identical seeding conditions, Caco-2 cell morphology on Corning MCs was evaluated on Days 4, 7, 10 and 14 (Figure 9).

Day 4 (Figure 9A): a single adherent cell was observed on the Corning MCs surface.

Day 7 (Figure 9B): a small, sparse cell cluster formed between microcarriers, notably smaller and less dense than cluster on PLA-Collagen MCs.

Day 10 (Figure 9C): cells showed signs of shrinkage and a collapsed membrane structure, suggesting reduced viability.

Day 14 (Figure 9D): no significant clusters development was observed; cells appeared morphological compromise, consistent with decreased viability.

Energy-dispersive X-ray spectroscopy analysis

EDS was performed on both microcarriers types to identify principal elemental compositions and distinguish between MCs and Caco-2 cells.

In both spectra (Figures 10 and 12), carbon and oxygen were the predominant elements in the microcarriers. Specifically, PLA-Collagen MCs contained 39.67% oxygen and 57.31% carbon, while Corning MCs contained 19.84% oxygen and 72.73% carbon.

Blank EDS maps of the PLA-Collagen and Corning scaffolds without seeding (Figures 11 and 13) show Carbon (C) and Oxygen (O), highlighted in red and green respectively, as the

main components of the scaffolds.

Carbon is present in the backbone formula of PLA (C₃H₄O₂)_n (Bahrami, Abenojar, & Martínez, 2020), the a carbon and carboxyl groups of the collagen amino acids (Wu, Cronin, & Crane, 2025), and the backbone formula of polystyrene (C₈H₈)_n (Gasusephol & Niebner, 2001), the main component of Corning Microcarriers (Hervy, et al., 2014).

Oxygen is present in most of the functional groups of microcarriers materials like carboxyl (COOH) and hydroxyl (OH) groups (Bahrami, Abenojar, & Martínez, 2020) (Wu, Cronin, & Crane, 2025).

Carbon and Oxygen are present in the blank samples of the scaffolds, but are also present in biological samples like the structure of the Caco-2 cells (Cooper, 2000)

, therefore, Carbon and Oxygen cannot be used as an indicator of the biological samples, but as a differentiator between the Caco-2 cells and the ECM.

Sodium (Na), a key indicator of Caco-2 cells due to transepithelial transport mechanism (Vieira-Coelho & Soares-da-Silva, 2001), served as the primary marker to differentiate

cellular regions from scaffolds. The EDS maps (Figures 14, 15 and 16 for PLA-Collagen MCs; Figures 16, 17 and 19 for Corning MCs) highlight sodium in yellow, providing a spatial representation of cell clusters over time.

PLA-Collagen MCs: a small, low-density cluster was noted on Day 3 (Figure 14D). An increase in cluster density followed on Day 7 (Figure 15D), consistent with SEM observations and viability data. By Day 14 (Figure 16D), large, densely populated clusters were apparent.

The same Elemental Analysis was performed for the commercial MCs (Corning MCs) and Caco-2 cell culture. For this group: on Day 4, a single cell was detected (Figure 17D). By day 7 (Figure 18D), a small cluster formed, through it remained sparse. On the final day (Figure 19D), no viable cluster was identified; only a morphologically compromised cell was seen.

Role of Porosity and Pore Size in Microcarrier-Cell Interactions

Our comparative analysis of PLA-collagen microcarriers (MCs) and commercially Corning collagen-coated MCs revealed key differences in size porosity and surface morphology that collectively influence cell-scaffold interactions. Specifically, the average diameter of PLA MCs was 45.6 ± 2.3 µm, markedly smaller than the 171.1 ± 2.1 µm diameter measure for the commercial MCs. This discrepancy aligns with the variability in MCs dimensions reported in the literature. For example, Li et al. (Li, et al., 2018) described microcarriers with an average diameter of 291.9 ± 30.7 µm, while Palazzo et al. (Palazzo, et al., 2021) reported PLA/PLGA MCs ranging from 0.4 ± 0.09 µm to 3 ± 0.9 µm. Such variability underscores the importance of tailoring microcarrier attributes -particularly size- to the requirements of each specific cell type and experimental objective, ensuring optimal adhesion and proliferation.

In term of oxygen delivery, Preissmann et al. (Preissmann, Wiesmann, Buchholz, Werner, & Noé, 1997) reported that oxygen does not generally exceed 300 µm in tissue. Given that Caco-2 cells range from 5 to 100 µm in size, depending on tumor stage and subtype (Granados-Romero, et al., 2017), both PLA-collagen MCs and Corning MCs fall within an acceptable range to avoid severe oxygen limitations. However, the relatively larger size of the commercial MCs places them closer to the upper threshold for oxygen diffusion, which could limit cellular conglomerate formation and nutrient exchange in higher-density cultures. Thus, such variation in MC size has direct implications for oxygen and nutrient diffusion.

Surface properties further distinguish PLA MCs from the reference commercial material. The PLA MCs featured a rough, highly porous topography with interconnected pores, increasingly recognized for their role as a key determinant of cellular behavior. Indeed, previous studies suggest a strong relationship between surface topology and cell adhesion. For example, polymer demixing can produce “nanoislands” that enhance cell adhesion if below 20 nm, whereas larger profusion can reduce adhesion (Kearns, McMurray, & Dalby, 2011). Such topographical cues promote focal adhesion formation, aiding cell attachment and proliferation. The enhanced roughness of PLA MCs thus provides an expanded surface area for cell-material interaction.

Pore formation is another critical factor influencing cell-microcarrier interactions. Large pore size typically facilitates greater cell infiltration and adhesion (Loh & Choong, 2013), but pores that are too small may impede nutrient and oxygen diffusion, whereas excessively large pores may cause cell loss (Gao, Yu, Wang, He, & Ding, 2022). The optimal balance between pore size and porosity thus emerges as crucial for maximizing growth while preserving mechanical integrity. Porosity levels as high as 96.7% have been reported to improve MSC proliferation through enhanced nutrient and oxygen exchange (Loh & Choong, 2013). Moreover, the interconnectivity of pores (on the order of (~120 µm) can further promote fibrovascular ingrowth by increasing surface area and scaffold degradation rates (Lutzweiler, Ndreu-Halili, & Engin-Vrana, 2020). These topographical features can engage various signaling pathways, such as Rho/ROCK and MAPK, which modulate differentiation. For instance, RhoA signaling promotes osteogenesis, while substrate stiffness can guide MSC lineage specification into neural, myogenic, or osteogenic cell types (Kearns, McMurray, & Dalby, 2011; Gao, Yu, Wang, He, & Ding, 2022). Similarly, collagen- and gelatine-based MCs have been shown to reinforce MSC osteogenesis via cytoskeletal rearrangements and mechanotransduction pathways (Badenes, et al., 2016). By carefully tuning particle diameter, porosity, interconnectivity and surface topography, microcarriers can be engineered to emulate large-scale tissue features, including axonal pathways or vascular channels exceeding 300 µm (Kearns, McMurray, & Dalby, 2011). These characteristics underscore the potential of PLA MCs for applications involving complex tissue architectures (Loh & Choong, 2013).

Finally, incorporating bioactive molecules into third-generation PLA MCs could further expand their utility by enabling controlled release of growth factors. However, achieving precise spatiotemporal delivery remains a notable challenge in tissue engineering (Lutzweiler, Ndreu-Halili, & Engin-Vrana, 2020).

Viability Monitoring and Proliferation Dynamics on Collagen-PLA Microcarriers

The unique porosity and collagen coating on the PLA MCs substantially improved Caco-2 cells adhesion compared to commercial alternatives. Collagen´s interaction with cell-surface integrins likely accounts for this enhanced performance, as it closely recapitulates the basal lamina of epithelial tissues (Jakob, et al., 2016). Additionally, collagen may bind growth factors secreted by the cells (Ragetly, Griffon, & Chung, 2010; He, Ma, Yong, Teo, & Ramakrishna, 2015), thereby bolstering intercellular communication and promoting the marked rise in cell density observed by Day 7.

The experimental design focused on quantifying only viable, microcarrier-adherent cells, ensuring a direct measure of the MCs effectiveness in fostering stable cellular attachment and growth. Notably, the decrease in cell number on Day 1 is consistent with the early adhesion phase typical of epithelial and other anchorage-dependent cell types (Hofmann, et al., 2007). Cells failing to adhere often undergo programmed cell death, highlighting the critical importance of initial attachment.

The CMFDA labelling result, along with the evidence of large cell conglomerates (confirmed by DAPI staining and bright field images), confirmed that viable cells continued to proliferate on collagen-PLA MCs, surpassing the performance of the commercial Corning MCs. These findings align with the work of Zhou et al. (Zhou, Ye, Zhou, & Tan, 2019), who reported a 3.25-fold increase in chondrocyte density on a polylactic acid (PLA) matrix relative to a 1.26-fold increase on commercial scaffolds. Collectively, these results affirm the value of collagen-PLA MCs for supporting sustained cellular expansion and three-dimensional growth.

Furthermore, the arrangement of epithelial cells on the microcarriers suggest that this system could approximate physiological conditions (Sensi, D’Angelo, D’Aronco, Molinaro, & Agostini, 2019) (Herrmann, et al., 2014). This property is highly relevant for disease modelling, where maintaining an environment that closely mimics the in vivo microenvironment is paramount for translational applicability.

Characterization of Microcarrier Structure and Caco-2 Cell Interaction

SEM images provided detailed insight into MCs structure, verifying that Caco-2 cells preferentially adhered to the MC surface without penetrating the pores. This is characteristic of microporous MCs, which feature pores too small for cell infiltration but sufficient to enhance nutrient, metabolite and gas exchange (Nogueira, Rodrigues, & Cabral, 2021; Kalasar & Alshomer, 2016). In contrast, macroporous MCs contain larger pores that allow cells to populate their interior, leading to distinct functional properties (Newland, et al., 2019).

The microporous structure of PLA-collagen MCs facilitates rapid cell attachment. By Day 3, individual cells adhered to the scaffold´s surface, as confirmed by the CMFDA assay indicating active metabolism and cell division (Lulevich, Shih, Lo, & Liu, 2010). Cell clusters emerged by Day 7, coinciding with peak proliferation as evidenced by both microscopy and growth kinetics data. By day 14, confluent cell assemblies covered the microcarriers, exhibited a roughened surface indicative of early tissue-like organization. Sinnecker et al. (Sinnecker, Ramaker, K, & Frey, 2014) reported that Caco-2 cells become more enterocyte-like by Day 14 of confluence, forming aggregate without fully developed brush border structures (Akeel, 2018).

The small intestine comprises a single layer of predominantly enterocytes, which display a brush border of microvilli for nutrient uptake (Schimpel, et al., 2015). The rough surface observed in Figure 8E and 8F likely indicate preliminary enterocyte-like differentiation and brush border formation (Akeel, 2018), roughness in the cytoskeletal structure of the cellular cluster could indicate the formation of cytoskeletal brush border proteins (Peterson & Mooseker, 1992).

By contrast, the commercial Corning® MCs supported only minimal cell attachment and viability over 14 days, with small cluster formation at Day 7 but little additional growth thereafter. Cells exhibited morphological features of comprised viability, emphasizing the importance of matching ECM composition and scaffold design to specific cell types (Zhang, Wang, Hui, Qiu, & Wang, 2017). In this regard, the PLA-collagen MCs functioned effectively by providing an ECM-like environment conducive to Caco-2 adhesion and proliferation.

Elemental Composition of PLA-Collagen Microcarriers

EDS confirmed that carbon (57.31%) and oxygen (39.67%) were the primary constituents of PLA-collagen MCs, aligning with the work of Kudzin et al. (Kudzin & Mrozinska, 2020), who found 51.7% carbon and 48.33% oxygen in biofunctionalized PLA materials. The slight compositional difference observed here likely stems from the collagen treatment, as collagen itself contains a substantial fraction of amino acids, particularly glycine, hydroxyproline and proline, which together comprise approximately 57% of collagen’s amino acid pool (Gauza-Wlodarczyk, Kubisz, & Wlodarczyk, 2017).

EDS Analysis of Caco-2 Cells

Sodium was selected as an elemental marker for Caco-2 cells, owing to its role in transepithelial transport via the Na+/K+ ATPase, which is critical for cellular homeostasis (Vieira-Coelho & Soares-da-Silva, 2001). EDS mapping corroborated the presence of viable cell clusters, in agreement with SEM observations, epifluorescence data and growth kinetics. Notably, enterocyte-like differentiation did not impede the detection of sodium, as Na+/K+ ATPase actively remains essential in mature enterocytes (Thorsen, Drengstig, & Ruoff, 2014). These convergent analytical findings underscore the suitability of PLA-collagen MCs to support Caco-2 cell adhesion, proliferation and differentiation, thereby positioning them as a robust platform for advanced intestinal tissue modelling.

In summary, PLA-collagen microcarriers demonstrate a clear advantage over commercial standards by providing a robust, reproducible platform for 3D cell culture. Their porous architecture and bioactive surface support efficient cell adhesion, proliferation and dense conglomerate formation -key prerequisite for complex tissue engineering, organoid development and disease modelling. Enhanced scalability further underscores their suitability for large-scale applications, including high-throughput screening and drug discovery. Future studies aiming to incorporate bioactive molecules, growth factors, or co-culture designs promise to expand the utility of PLA-collagen MCs, offering a versatile and powerful tool for advancing both fundamental research and translational applications in regenerative medicine and oncology.

Conflicts of interest

There are no conflicts of interest to declare.

Author Contribution

A.P.S.-U., I.S.-C., N.R.-Z., S.N.-N., N.F.D., D.A., D.M.-A., and N.E.D.-M. contributed to the conception and design of the study, data acquisition, and analysis. A.P.S.-U., I.S.-C., and N.R.-Z. wrote the main text of the manuscript. S.N.-N. and D.A. prepared the figures. N.F.D. and D.M.-A. critically revised the manuscript for intellectual content. N.E.D.-M. (Néstor Emmanuel Díaz Martínez) supervised and coordinated the project, handled correspondence, and handled administrative/financial arrangements related to the study. All authors reviewed and approved the final version of the manuscript. N.E.D.-M. is the corresponding author.

Acknowledgement

The authors gratefully acknowledge the Nanotechnology Laboratory of the Instituto Tecnológico y de Estudios Superiores de Occidente (ITESO) for providing access to their facilities and supporting the SEM analysis, which was instrumental in the completion of this study. And grant CBF2023-2024-4914.

Akeel, M. (2018). Endocytic pathway in Caco-2 cells: A model for drugs transport across the epithelial cell barrier. Edorium J Gastroenterol, 5.
Al Shehadat, S., Gorduysus, M., Hamid, S., Abdullah, N., Samsudin, A., & Ahmad, A. (2018). Optimization of scanning electron microscope techniue for amniotic membrane investigation: A preliminary study. European Journal of Dentistry, 12(4), 574-578.
Arneth, B. (2019). Tumor Microenvironment. Medicina(Kanuas), 1(15), 30-;56.
Badenes, S. M., Fernandes-Platzgummer, A., Rodrigues, C. A., Diogo, M. M., Da Silva, C. L., & Cabral, J. M. (2016). Microcarrier Culture Systems for Stem Cell Manufacturing. In Stem Cell Manufacturing (pp. 77-104). Elsevier.
Bahrami, M., Abenojar, J., & Martínez, M. A. (2020). Recent progress in hybrid biocomposites: Mechanical properties, water absorption, and flame retardancy. materials, 13.
Bello, A. B., Kim, D., Kim, D., Park, H., & Lee, S. H. (2020). Engineering and functionalization of gelatin biomaterials: From cell culture to medical applications. Tissue Engineering Part B: Reviews, 26(2), 164-180.
Bergenheim, F., Seidelin, J. B., Pedersen, M. T., Karp, J., Nielsen, O., Mead, B., & Jensen, K. (2019). Fluorescence-based tracing of transplanted intestinal epithelial cells using confocal laser endomicroscopy. Stem Cell Res Ther, 10, 148.
Bhattacharjee, Y. (2012). Pharma firms push for sharing of cancer trial data. Science, 338, 29.
Boursier, J. F., Fournet, A., Bassanino, J., Manassero, M., Bedu, A. S., & Leperlier, D. (2018). Reproducibility, accuracy and effect of autoclave sterilization on a thermoplastic three-dimensional model printed by a desktop fused deposition modelling three-dimensional printer. Veterinary and Comparative Orthopaedics and Traumatology, 31(06), 422-430.
Cawkill, D., & Eaglestone, S. S. (2007). Evolution of cell-based reagent provision. Drug discovery today, 12, 820-825.
Chakraborty, R., Asthana, A., Singh, A., Adhikari, R., & Hasan, M. (2023). Chapter 20 - Collagen - a highly developed and abundant fibrous protein: synthesis and characterization. Handbook of Natural Polymers, 1(20), 489-508.
Chen, A. K., Chen, X., Choo, A. B., Reuveny, S., & Oh, S. K. (2011). Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem cell research, 7(2), 97-111.
Chen, X., Zhou, L., Xu, H., Yamamoto, M., Shinoda, M., Kishimoto, M., . . . Yamane, H. (2020). Effect of the Application of a Dehydrothermal Treatment on the Structure and the Mechanical Properties of Collagen Film. Materials, 13(2), 377.
Convention, U. S. (2018). <USP71> Sterility tests. In The United States Pharmacopeia 2018: USP 41 - NF 36 (pp. 5984-5991). Rockville: United States Pharmacopeial Convention.
Cooper, G. M. (2000). The Cell: A Molecular Approach. Sunderland (MA): Sinauer Associates.
Costa, R. C., Pereira, E. D., Silva, F. M., Jesus, E. O., & Souza Jr, F. G. (2018). Drug micro-carriers based on polymers and their sterilization. Chemistry & Chemical Technology, 12, 473-487.
Cui, M., Liu, L., Guo, N., Su, R., & Ma, F. (2015). Preparation, cell compatibility and degradability of collagen-modified poly (lactic acid). Molecules, 20(1), 595-607.
Derakhti, S., Safiabadi-Tali, S. H., Amoabediny, G., & Sheikhpour, M. (2019). Attachment and detachment strategies in microcarrier-based cell culture technology: A comprehensive review. Materials Science and Engineering: C, 103, 109782.
Europe, C. o. (2018). Chapter 2.6.1: Sterility. In European Pharmacopoeia. Strasbourg: Council of Europe.
Farid, S. S., & Jenkins, M. J. (2018). Chapter 44: Bioprocesses for cell therapies. In Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes (pp. 899-930). Elsevier.
Freshney, R. I. (2015). Culture of animal cells: a manual of basic technique and specialized applications. Hoboken, NJ: John Wiley & Sons.
Gao, J., Yu, X., Wang, X., He, Y., & Ding, J. (2022). Biomaterial–related cell microenvironment in tissue engineering and regenerative medicine. Engineering, 13, 31-45.
Gasusephol, B., & Niebner, N. (2001). Polystrene and Styrene Coploymers. Encyclopedia of Materils: Science and Technology, 2, 7735-7741.
Gauza-Wlodarczyk, M., Kubisz, L., & Wlodarczyk, D. (2017). Amino acid composition in determination of collagen origin and assesment of physical factors effects. International Journal of Biological Macromolecules, 104, 987-991.
Ghosh, S., Spagnoli, G. C., Martin, I., Ploegert, S., Demougin, P., Heberer, M., & Reschner, A. (2005). Three‐dimensional culture of melanoma cells profoundly affects gene expression profile: A high density oligonucleotide array study. Journal of cellular physiology, 204, 522-531.
Granados-Romero, J. J., Valderrama-Treviño, A. I., Contreras-Flores, E. H., Barrera-Mera, B., Herrera-Enríquez, M., Uriarte-Ruíz, K., & Arauz-Peña, G. (2017). Colorectal cancer: A review. Int J Res Med Sci, 5(11), 4667.
Griffith, L. G., & Swartz, M. A. (2006). Capturing complex 3D tissue physiology in vitro. Nature reviews Molecular cell biology, 7(3), 211-224.
Gupta, B., Semwal, V., & Pathak, A. (2020). Chapter 7 - Nanotechnology-based fiber-optic chemical biosensors. Nano-Optics, 163-19.
Handral, H. K., Wyrobnik, T. A., & Lam, A. L. (2023). Emerging trends in biodegradable microcarriers for therapeutic applications. Polymers, 15(6), 1487.
Haycock, J. (2011). 3D Cell Culture: A review of Current Approaches. Humana Press, 1, 15.
He, W., Ma, Z., Yong, T., Teo, W. E., & Ramakrishna, S. (2015). Fabrication of collagen-coated biodegradable polymer nanofiber mesh and its potential for endothelial cells growth. Biomaterials, 26(36), 7606–7615. doi:10.1016/j.biomaterials.2005.05.049
Herrmann, D., Conway, J. R., Vennin, C., Magenau, A., Hughes, W. E., Morton, J. P., & Timpson, P. (2014). Three-dimensional cancer models mimic cell–matrix interactions in the tumour microenvironment. Carcinogenesis, 35(8), 1671-1679. doi:https://doi.org/10.1093/carcin/bgu108
Hervy, M., Weber, J., Pecheul, M., Dolley-Sonneville, P., Henry, D., Zhoy, Y., & Melkoumian, Z. (2014). Long Term Expansion of Bone Marrow-Derived hMSCs on Novel Synthetic Microcarriers in Xeno-Free, Defined Conditions. PLoS One, 9(3). Retrieved 02 05, 2025, from https://www.corning.com/catalog/cls/documents/product-information-sheets/CLS-CC-075-A4.pdf
Heu, R., Shahbazmohamadi, S., & Yorston, J. (2019). Target Material Selection for Sputter Coating of SEM Samples. Microscopy Today, 27(4), 32-36.
Hofmann, C., Obermeier, F., Artinger, M., Hausmann, M., Falk, W., Schoelmerich, J., . . . Grossmann, J. (2007). Cell-cell contacts prevent anoikis in primary human colonic epithelial cells. Gastroenterology, 132(2), 587-600. doi:10.1053/j.gastro.2006.11.017
Hong, Y., Gao, C., Xie, Y., Gong, Y., & Shen, J. (2005). Collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials, 26(32), 6305-6313.
Hu, M., Li, Y., Huang, J., Wang, X., & Han, J. (2021). Electrospun scaffold for biomimic culture of Caco-2 cell monolayer as an in vitro intestinal model. ACS Applied Bio Materials, 4(2), 1340-1349.
Hurni, M., Noach, A., Blom-Roosemalen, M., de Boer, A., Nagelerke, J., & Breimer, D. (1993). Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrousidate: assesment of effect-reversibility and imaging of transepithelial transport routes by confocal laser scanning microscopy. J Pharmacol Ex Ther., 267(2), 942-950.
Jakob, P. H., Kehrer, J., Flood, P., Wiegel, C., Haselmann, U., Meissner, M., . . . Reynaud, E. G. (2016). A 3-D cell culture system to study epithelia functions using microcarriers. Cytotechnology, 68, 1813-1825. doi: 10.1007/s10616-015-9935-0
Kalasar, D., & Alshomer, F. (2016). Capítulo 8 - Señales micro y nanotopográficas que guían la respuesta del huésped al nanomaterial. In situ Tissue Regeneration, 137-163.
Kapałczyńska, M., Kolenda, T., Przybyła, W., Zajączkowska, M., Teresiak, A., Filas, V., . . . Lamperska, K. (2018). 2D and 3D cell cultures–a comparison of different types of cancer cell cultures. Archives of Medical Science, 14(4), 910-919.
Karak, N. (2019). Chapter 2. Silver Nanomaterials and Their Polymer Nanocomposites. Nanomaterials and Polymer Nanocomposites(2), 47-89.
Kawai, S., Yamazaki, M., Shibuya, K., Yamazaki, M., Fujii, E., Nakano, K., & Suzuki, M. (2020). Three-dimensional culture models mimic colon cancer heterogeneity induced by different microenvironments. Scientific Reports, 10(1), 3156.
Kearns, V. R., McMurray, R. J., & Dalby, M. J. (2011). Biomaterial surface topography to control cellular response: technologies, cell behaviour and biomedical applications. In Surface Modification of Biomaterials (pp. 169-201). Woodhead Publishing.
Keles, H., Naylor, A., Clegg, F., & Sammon, C. (2015). Investigation of factors influencing the hydrolytic degradation of single PLGA microparticles. Polymer degradation and stability, 119, 228-241.
Kohn, J., Welsh, W. J., & Knight, D. (2007). A new approach to the rationale discovery of polymeric. Biomaterials, 28, 4171-4177.
Kudzin, M., & Mrozinska, Z. (2020). Biofunctionalization of Textile Materials. 3. Fabrication of Poly(lactide-Potassium Iodide Composites with Antifungal Properties. Coatings, 3, 92-103.
Kumar, S., Balaji, D., & Mandlimath, T. (2023). 3 - Characterization of flexible ceramics. Advanced Flexibe Ceramics, 25-43.
Lea, T. (2015). Chapter 10: Caco-2 Cell Line. In Caco-2 cell line. The Impact of Food Bioactives on Health: in vitro and ex vivo models (pp. 103-111). The Netherlands: SpringerOpen.
Lee, J., Cuddigy , M. J., & Kotov, N. A. (2008). Three-dimensional cell culture matrices: state of the art. Tissue engineering part B: reviews, 14(1), 61-86.
Leon-Mancilla, B., Araiza-Téllez, M., Flores-Flores, J., & Pia-Barba, M. (2015). Physico-chemical characterization of collagen scaffolds for tissue engineering. Journal of Applied Research and Technology, 14, 77-85.
Li, L., Song, K., Chen, Y., Wang, Y., Shi, F., Nie, Y., & Liu, T. (2018). Design and biophysical characterization of poly(l-lactic) acid microcarriers with and without modification of chitosan and nanohydroxyapatite. Polymers, 10(10), 1061.
Loh, Q. L., & Choong, C. (2013). Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng Part B Rev, 19(6), 485-502.
Lulevich, V., Shih, Y., Lo, S., & Liu, G. (2010). Cell tracing dyes significantly change single cell mechanics. J Phys Chem B, 113(18), 6511-6519.
Lutzweiler, G., Ndreu-Halili, A., & Engin-Vrana, N. (2020). The overview of porous, bioactive scaffolds as instructive biomaterials for tissue regeneration and their clinical translation. Pharmaceutics, 12(7), 602.
Muschler, G. F., Nakamoto, C., & Griffith , L. G. (2004). Engineering principles of clinical cell-based tissue engineering. JBJS, 86(7), 1541-1558.
Nath, S. C., Harper, L., & Rancourt, D. E. (2020). Cell-based therapy manufacturing in stirred suspension bioreactor: thoughts for cGMP compliance. Frontiers in Bioengineering and Biotechnology, 8, 599674.
Newland, B., Ehret, F., Hoppe, F., Eigel, D., Pette, D., Newland, H., . . . Werner, C. (2019). Macroporous heparin-based microcarriers allow long-term 3D culture and differentiation of neural precursor cells. Biomaterials.
Nogueira, D., Rodrigues, C., & Cabral, C. (2021). Chapter 7 - Emerging strategies for scalable human iduced pluripotent stem cell expansion and differentiation. Methods in iPSC Technology, 9, 163-185.
Palazzo, I., Lamparelli, E. P., Ciardulli, M. C., Scala, P., Reverchon, E., Forsyth, N., & Della-Porta, G. (2021). Supercritical emulsion extraction fabricated PLA/PLGA micro/nano carriers for growth factor delivery: Release profiles and cytotoxicity. International Journal of Pharmaceutics, 592, 120108.
Pampaloni, F., Reynaud, E. G., & Stelzer , E. H. (2007). The third dimension bridges the gap between cell culture and live tissue. Nature reviews Molecular cell biology, 8(10), 839-845.
Peterson, M., & Mooseker, M. (1992). Characterization of enterocyte-like brush border cytoseleton of the C2BBe, clones of the human intestinal cell line, Caco-2. Journal of Cell Science, 102(3), 581-600.
Piccinini, F., Tesei, A., Arienti, C., & B. A. (2017). Cell counting and viability assessment of 2D and 3D cell cultures: expected reliability of the trypan blue assay. Biological procedures online, 19, 1-12.
Preissmann, A., Wiesmann, R., Buchholz, R., Werner, R. G., & Noé, W. (1997). Investigations on oxygen limitations of adherent cells growing on macroporous microcarriers. Cytotechnology, 24, 121-134.
Ragetly, G., Griffon, D. J., & Chung, Y. S. (2010). The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis. Acta Biomaterialia, 6(10), 3988–3997. doi:10.1016/j.actbio.2010.05.016
Rodríguez-Herrero, Y., Lopez-Camas, & Ullah, A. (2023). Chapter 4 - Characterization of biobased materials. Advanced Applications of Biobased Materials, 111-143.
Samal, R., Gautam, D., Panmei, K., Lanbiliu, P., Saya, L., Gambhir, G., . . . Kumar, S. (2024). Evolution in graphene oxide-based materials characterization and modeling. Comprehensive Materials Processing, 2(16), 210-220.
Santoro, M., Shah, S. R., Walker, J. L., & Mikos, A. G. (2016). Poly (lactic acid) nanofibrous scaffolds for tissue engineering. Advanced drug delivery reviews, 107, 206-212.
Schimpel, C., Werzer, O., Fröhlich, E., Leitinger, G., M, A.-N., Teubl, B., . . . Roblegg, E. (2015). Atomic force microscopy as analytical tool to study physico.mechanical properties of intestinal cells. Beilstein Journal of Nanotechnology, 6, 1457-1466.
Sehgal, P. K., & Srinivasan, A. (2009). Collagen-coated microparticles in drug delivery. Expert opinion on drug delivery, 6(7), 687-695.
Sensi, F., D’Angelo, E., D’Aronco, S., Molinaro, R., & Agostini, M. (2019). Preclinical three‐dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation. Journal of cellular physiology, 234(1), 181-191. doi:10.1002/jcp.26812
Shojaei, T., Soltani, S., & Derakhshani, M. (2022). Chapter 6 - Synthesis, properties, and biomedical applications of inorganic materials. Fundamentals of Bionanometerials, 139-174.
Sinnecker, H., Ramaker, K, & Frey, A. (2014). Coating with luminal gut-constituents alters adherence of nanoparticles to intestinal epithelial cells. Beilstein Journl of Nanotechnology, 5, 2308-2315.
Smietana, K., Siatkowski, M., & Moller, M. (2016). Trends in clinical success rates. Nature Reviews. Drug discovery, 15, 379-380.
Stratton, S., Shelke, N. B., Hoshino, K., Rudraiah, S., & Kumbar, S. G. (2016). Bioactive polymeric scaffolds for tissue engineering. Bioactive materials, 1(2), 93-108.
Thavarajah, R., Mudimbaimannar, V., Elizabeth, J., Rao, U., & Ranganathan, K. (2012). Chemical and physical basics of routine formaldehyde fixation. Journal of Oral and Maxilofacial Pathology, 16(3), 400-405.
Thorsen, K., Drengstig, T., & Ruoff, P. (2014). Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model. Am J Physiol Cell Physiol, 307(4), 320-337.
Urwyler, P., & Schift, H. (2016). Chapter 14: Nanostructured Polymers for Medical Applications. In Nanoscience and Nanotechnology for Human Health (pp. 293-314). Wiley.
Vieira-Coelho, M., & Soares-da-Silva, P. (2001). Comparatie study on sodium transport and Na+/K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine. Life Sci., 69(17), 1969-1981.
Weadock, K. S., Miller, E. J., Bellincampi, L. D., Zawadsky, J. P., & Dunn, M. G. (1995). Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment. Journal of biomedical materials research, 29(11), 1373-1379.
Wu, M., Cronin, K., & Crane, J. S. (2025). Biochemistry, Collagen Synthesis. Tresure Island (FL): StatPearls Publishing.
Xiao, K., Xu, Y., Cao, X., Xu, H., & Li, Y. (2022). Chapter 18. - Advanced characterization of membrane surface fouling. 60 Years of the Loeb-Sourirajan Membrane, 499-532.
Xiao, W., Ehsanipour, A., Sohrabi, A., & Seidlits, S. K. (2018). Hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells. Journal of Visualized Experiments: JoVE.
Xiao, Y., Xu, Y., Lu, J., Zhu, X., Fan, H., & Zhang, X. (2007). Preparation and characterization of collagen-modified polylactide microparticles. Materials Letters, 61(13), 2601-2605.
Yamada, K. M., & Cukierman, E. (2007). Modeling tissue morphogenesis and cancer in 3D. Cell, 130, 601-610.
Yin, S., & Cao, Y. (2021). Hydrogels for large-scale expansion of stem cells. Acta biomaterialia, 128, 1-20.
Zhang, Z., Wang, B., Hui, D., Qiu, J., & Wang, S. (2017). D bioprinting of soft materials-based regenerative vascular structures and tissues. Composites Part B: Engineering, 123, 279-291.
Zhou, A., Ye, Z., Zhou, Y., & Tan, W. (2019). Bioactive poly(ε-caprolactone) microspheres with tunable open pores as microcarriers for tissue regeneration. Journal of Biomaterials Applications, 33(9), 1242-1251. doi:10.1177/0885328218825371
Zhou, N., Liu, Y. D., Zhang, Y., Gu, T. W., & Peng, L. H. (2023). Pharmacological functions, synthesis, and delivery progress for collagen as biodrug and biomaterial. Pharmaceutics, 15(5), 1443.
Zhou, W., Kang, H., O'Grady, M., Chambers, K. M., Dubbels, B., Melquist, P., & Gee, K. R. (2016). CellTrace™ far red & CellTracker™ Deep red. Journal of Biological Methods, 3, 38.

No competing interests reported.

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Engineering Biocompatible Polylactic Acid (PLA) Microcarriers for Enhanced 3D Cell Culture

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