Electrospun cell culture and tissue scaffolds: materials, structures, and research progress

01Cell culture substrates and three-dimensional scaffolds: first clarify what needs to be observed

When developing cell culture materials, the experimental question must first be clarified: is the goal to improve initial adhesion, observe alignment and migration, or enable cells to enter a structure of a certain thickness and deposit extracellular matrix? These goals correspond to different requirements for porosity, support, and surface properties. Results from conventional two-dimensional culture should be evaluated separately from cell distribution within three-dimensional scaffolds; a scaffold cannot be said to achieve three-dimensional culture merely because it has thickness in appearance.^3,4,5,6

“Universal tissue scaffold” here refers to a material platform that can be adjusted for different cells and experimental purposes; it does not mean that a single formulation is suitable for all tissues. Researchers must still specify the cell source, culture conditions, culture duration, and the functions they wish to preserve or induce.^{1,2,3,4,5,6,7}

02From materials to pore structures: which variables can be controlled by electrospinning

Electrospinning can produce fibers from synthetic polymers combined with components such as proteins, or it can first create a skeleton followed by surface immobilization or post-treatment. Blending alters not only composition but also solution state, fiber dimensions, and wettability; therefore, when comparing two formulations, structural changes should be analyzed together with cellular outcomes.^1,2,3,7

Structurally, fiber diameter, random or ordered arrangements, coexistence of fibers at different scales, and layer-by-layer stacking of grids can be discussed. Finer fibers promote abundant contact interfaces, but dense packing may restrict cell penetration into the interior. Scaffold design must identify a combination of contact area, porosity, support, and cell behavior appropriate for the specific project.^3,4,5

03Cell-adhesion interfaces: protein blending and post-modification of collagen

Wool keratin blended with PCL

Wu et al. (2018) extracted and regenerated keratin from wool, electrospun it in blends with PCL, and compared wettability, degradation, and cell culture performance across different ratios. A 7-day mouse fibroblast experiment showed that keratin-containing systems improved cell adhesion and viability. This indicates that natural proteins can serve as components to modulate the contact interface of synthetic polymers, without needing to independently fulfill fiber-forming and structural-support functions.¹

The study’s results pertain to a specific keratin-processing method, the PCL system, and the cell model used. After changing the raw-material source or extraction process, the formulation’s fiber-forming and cellular performance must be revalidated; “natural origin” cannot be equated with good biocompatibility under all application conditions.¹

First electrospin the PVA skeleton, then attach collagen

A Mexican team (Pérez-Nava et al., 2023) adopted a different approach: they first prepared polyvinyl alcohol (PVA) nanofibers and subsequently immobilized collagen on the surface through further reactions. Combining chemical characterization, mechanical testing in both dry and wet states, and HEK-293 cell culture, they observed improved cytocompatibility after surface functionalization. This demonstrates that biological components need not necessarily undergo the same spinning solvent and fiber-forming process; they can also be introduced after the skeleton has been formed.²

For sample development, this means that “how to stabilize film formation” and “which surface cells encounter” can be separated into two independently comparable variables. During post-modification, the degree of bonding, washing effects, and wet-state retention should also be tracked, rather than assuming that a stable functional layer has formed on the surface merely because collagen was added to the solution.²

04Enabling cell penetration into the interior: multiscale fibers, direct writing, and removable components

Micrometer-scale fibers provide space; nanometer-scale fibers provide contact

Li et al. (2016) used needleless disc electrospinning to compare the nanoscale structure of PCL/ gelatin with its multiscale micro–nano structure. The multiscale scaffold exhibited larger pores, and culture of mouse fibroblasts and rat bone marrow stromal cells showed improved proliferation and internal penetration. This study separated “fiber uniformity” from “suitability of the culture structure”: not all applications require uniformly fine fibers.³

Controlling pore positions through near-field direct writing

He et al. (2018) used near-field electrospinning to directly write and layer-stack grids, constructing a PCL/ hydroxyapatite scaffold with a reported average pore diameter of approximately 167 micrometers. Mouse pre-osteoblasts MC3T3-E1 were able to adhere, proliferate, and exhibit differentiation-related changes within the three-dimensional structure. This provides a method for culture scaffolds requiring describable pore layouts, although this pore size is not a universal standard for all cells.⁴

Removing selected fibers to create channels for internal penetration

Baker et al. (2012) collected slowly degradable PCL fibers together with soluble polyethylene oxide (PEO) fibers, then removed PEO. Culture of human meniscus-derived cells showed that increasing the removable component improved internal cell and matrix distribution, while the retained aligned fibers continued to provide directional cues. The study also noted that cell traction could deform looser structures; therefore, boundary fixation was used for long-term culture. In vitro results from human cells do not represent outcomes of patient implantation.⁵

05Three-dimensional shape and wet-state stability: placing samples into the actual culture process

Gao et al. (2018) placed electrospun membranes into 3D printed molds and used a foaming process to form porous, layered scaffolds with predefined shapes, followed by in vitro cell evaluation. They also used bioprinting to position cells initially and observed their migration from hydrogels into the scaffolds. This demonstrates a combination of electrospinning, molding, and cell-patterning methods suitable for inspiring culture designs that require both specific shapes and spatial distributions.⁶

Gu et al. (2024) focused on a more practical issue: shrinkage of PLGA fiber scaffolds after entering the culture environment. They incorporated PCL and silk fibroin into PLGA and separately examined dimensional stability, wettability, in vitro degradation, and cell proliferation. The results support improving wet-state dimensions and cellular responses through composite components, indicating that maintaining sample morphology is itself part of culture-platform development.⁷

These two studies address different needs: the former actively constructs three-dimensional geometries, whereas the latter reduces unintended deformations during use. When making a practical selection, one should first determine whether free deformation, fixation within a culture frame, or integration with other scaffolds is required, and then discuss collection and post-treatment methods.^6,7

06What does “effective” specifically mean in cell experiments?

Viability staining, metabolic assays, cell counts, penetration depth, and functional markers do not answer the same question. An increase in cell number may be advantageous for expansion studies, but if the goal is to maintain a specific phenotype, morphological, expression-based, or functional assessments should also be included. When comparing across studies, cell source, seeding density, culture duration, and controls should all be considered rather than ranking materials solely by a single proliferation percentage.^{1,2,3,4,5,6,7}

Human cell lines or cells derived from human tissues can still be used for in vitro studies; this fact alone does not justify referring to them as human clinical trials. The materials and culture results cited in this document primarily help identify structures and experimental variables worthy of investigation. They do not directly demonstrate the formation of implantable tissue, much less prove that organ function has been restored.^2,5,6

07From a single experimental membrane to comparable culture samples

When communicating fabrication requirements, one can first specify the cell type, intended use, and sample form: a disc to be placed in a culture well, an aligned membrane fixed within a frame, a grid-like scaffold, or a three-dimensional structure of a specified thickness. Then clarify fiber orientation, thickness or surface density, effective porosity, sampling location, and wet-state dimensions, so that equipment configuration serves detectable objectives.

Random membranes typically emphasize stable fluid supply and uniform cell collection; ordered arrangements require attention to collection methods and directional consistency; near-field direct writing requires attention to trajectories and interlayer stacking; surface functionalization or foamed structures also require post-treatment to be included in batch records. Control groups should alter only the key variable under investigation whenever possible, avoiding simultaneous changes to formulation, membrane thickness, and culture conditions that would render results uninterpretable.

Before formal cell experiments, confirm whether cleaning, residue removal, sterilization or disinfection, pre-wetting, and fixation steps alter the material. Preparing a larger area can facilitate batch cutting of samples, but the fiber structure and cellular response at different locations and across different batches must still be compared. The process repeatability of the equipment and the reproducibility of biological experiments need to be linked through this shared sample record.^2,3,4,5,6,7

08Common issues in the development of fibrous membranes for cell culture

Does finer fiber necessarily yield better cell culture results?

Not necessarily. Fine fibers can provide a contact interface, but a denser structure may hinder cell penetration into the interior. Studies of multiscale fibers and direct-write grids suggest that the structure should be selected according to the objective of surface culture or three-dimensional culture.^3,4,5

Must collagen or other proteins be added directly to the spinning solution?

Not necessarily. Blending and post-spinning surface modification represent two distinct approaches. The processing conditions of the protein, its fiber-forming ability, surface presentation, and wet-state stability must be considered to determine which approach is more suitable for the planned experiment.^1,2

Can ordinary electrospun membranes be directly referred to as three-dimensional culture scaffolds?

It should be observed whether cells penetrate through the thickness direction and whether a reproducible distribution and the intended behavior are formed internally. A thicker scaffold or the presence of pores in a micrograph alone cannot answer these questions.^3,4,5,6

References

The following are the original studies used in this review, independently summarized by TONGLI. The interpretation of the literature does not imply that the paper authors or research institutions have equipment procurement or cooperative relationships with TONGLI; the specific reading scope is described in each literature entry.

Bibliographic records retain the original publication language for accurate lookup.

1. Wu P, Dai X, Chen K, Li R, Xing Y. Fabrication of regenerated wool keratin/polycaprolactone nanofiber membranes for cell culture. International Journal of Biological Macromolecules. 2018;114:1168–1173.

   DOI：10.1016/j.ijbiomac.2018.03.157 ↗

   Scope of review: the complete original local text, with verification of the PubMed abstract and bibliographic record

2. Pérez-Nava A, Espino-Saldaña AE, Pereida-Jaramillo E, et al. Surface collagen functionalization of electrospun poly(vinyl alcohol) scaffold for tissue engineering. Process Biochemistry. 2023;126:1–14.

   DOI：10.1016/j.procbio.2022.12.022 ↗

   Scope of review: the complete original local text, with verification of the author's institutional publication record

3. Li D, Chen W, Sun B, et al. A comparison of nanoscale and multiscale PCL/gelatin scaffolds prepared by disc-electrospinning. Colloids and Surfaces B: Biointerfaces. 2016;146:632–641.

   DOI：10.1016/j.colsurfb.2016.07.009 ↗

   Scope of review: the complete original local text, with verification of the PubMed abstract and bibliographic record

4. He FL, Li DW, He J, et al. A novel layer-structured scaffold with large pore sizes suitable for 3D cell culture prepared by near-field electrospinning. Materials Science and Engineering C. 2018;86:18–27.

   DOI：10.1016/j.msec.2017.12.016 ↗

   Scope of review: the complete original local text, with verification of the PubMed abstract and bibliographic record

5. Baker BM, Shah RP, Silverstein AM, et al. Sacrificial nanofibrous composites provide instruction without impediment and enable functional tissue formation. Proceedings of the National Academy of Sciences of the USA. 2012;109:14176–14181.

   DOI：10.1073/pnas.1206962109 ↗

   Scope of review: the original paper abstract and results section in PMC; the in vitro culture section of meniscal source cells cited in this article

6. Gao Q, Gu H, Zhao P, et al. Fabrication of electrospun nanofibrous scaffolds with 3D controllable geometric shapes. Materials & Design. 2018;157:159–169.

   DOI：10.1016/j.matdes.2018.07.042 ↗

   Scope of review: the complete original local text, with verification of the journal abstract and bibliographic record

7. Gu P, Mao Y, Lu W, Chen W. Silk fibroin incorporated electrospun aliphatic polyester nanofiber scaffolds with excellent dimensional stability and cytocompatibility. European Polymer Journal. 2024;211:113039.

   DOI：10.1016/j.eurpolymj.2024.113039 ↗

   Scope of review: the complete original local text, with verification of the journal's original abstract and conclusions