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2D and 3D Culture Systems: Current Trends and Biomedical Applications

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A special issue of Pharmaceuticals (ISSN 1424-8247). This special issue belongs to the section "Biopharmaceuticals".

Deadline for manuscript submissions: closed (25 January 2026) | Viewed by 18011

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Special Issue Editors

Dr. Juliana Mara Serpeloni

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Department of General Biology, Center of Biological Sciences, State University of Londrina (UEL), Londrina 86057-970, Brazil
Interests: natural products; chemoprevention; cell culture systems; 3D culture; oncogenetic; cancer biomarkers
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Prof. Dr. Colus Ilce Mara

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Department of General Biology, Center of Biological Sciences, State University of Londrina (UEL), Londrina 86057-970, Brazil
Interests: cell culture systems; antimutagenesis; genotoxicity; oncogenetic; cancer biomarkers; epigenetic
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Dr. Diego Luís Ribeiro

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Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (ICB/USP), São Paulo 05508-000, Brazil
Interests: mutagenesis; DNA Repair; 3D cell culture; 3D spheroids; translesion synthesis

Special Issue Information

Dear Colleagues,

This Special Issue of Pharmaceuticals will address recent advances in 2D and 3D in vitro cultures, exploring current trends and innovative biomedical applications. We are seeking submissions that discuss the latest techniques and highlight the implications of these advances in biomedical research, including disease modeling (e.g., cancer), drug testing and screening, tissue morphology and engineering, and regenerative therapies. Emerging technologies driving the evolution of these culture systems, promoting a deeper understanding of cellular processes and opening new possibilities for clinical applications will also be explored. In summary, the issue provides a holistic and updated view of 2D and 3D in vitro cultures, consolidating essential information for researchers and professionals involved in biomedicine.

Interested researchers are invited to submit an original research paper and review articles for publication in this Special Issue. Articles employing 3D cell cultures and those comparing results between 2D and 3D models are of particular interest. Studies concerning spheroids obtained through ex vivo propagation from individual patients (organoids) are also encouraged.

Dr. Juliana Mara Serpeloni
Prof. Dr. Colus Ilce Mara
Dr. Diego Luís Ribeiro
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pharmaceuticals is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

spheroids
organoids
3D cultures
tissue engineering
disease modelling
regenerative medicine
cell-extracellular matrix interaction
drug screening

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Published Papers (9 papers)

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Research

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17 pages, 2236 KB

Open AccessArticle

Simvastatin Enhances Stem Cell Osteogenesis and Reduces Peri-Implant Bone Loss: An In Vitro and a Randomized Clinical Study

by Asmaa Saleh, Shereen N. Raafat, Sherihan Ahmed Sayed, Mohamed Shamel, Sherif Shafik El Bahnasy and Sara F. El Shafei

Pharmaceuticals 2026, 19(3), 368; https://doi.org/10.3390/ph19030368 - 26 Feb 2026

Viewed by 551

Abstract

Background: Despite extensive preclinical evidence that statins enhance osteogenesis and the widespread clinical use of platelet-rich fibrin (PRF), the clinical effectiveness of statin-incorporated PRF (SIM-PRF) in limiting peri-implant crestal bone loss remains insufficiently validated. Objectives: To address the mentioned gap, we integrated in vitro assays on human periodontal ligament stem cells (hPDLSCs) with a controlled clinical trial to test whether SIM-PRF reduces early and 12-month marginal bone loss versus PRF alone and PRF with bone graft. Methods: In vitro, cytotoxicity, migration and osteogenic differentiation were assessed, in addition to the effect on basal inflammatory markers. Clinically, 24 immediate-implant cases were randomized to receive PRF, PRF+SIM, or PRF+bone graft, with CBCT-based crestal bone change measured at 0–3, 3–6, and 6–12 months. Results: Flow cytometry confirmed the mesenchymal identity of the isolated hPDLSCs, which exhibited dose-dependent responses to SIM treatment. Lower SIM concentrations (0.1 μM) enhanced osteogenic differentiation, as evidenced by increased mineralization, alkaline phosphatase activity, and expression of osteogenic markers (RUNX2 and osteocalcin), while maintaining cell viability and migration. Both SIM concentrations (0.1 μM and 1 μM) significantly reduced basal pro-inflammatory cytokine expression (TNF-α and IL-6). Radiographic analysis revealed significantly reduced crestal bone loss (p < 0.001) in the PRF-SIM and PRF-Bone groups compared to PRF alone, particularly during early postoperative intervals (0–3 and 3–6 months). Notably, no significant difference was observed between the PRF-SIM and PRF-Bone groups (p > 0.05) in preserving the peri-implant bone. Conclusions: These findings highlight the potential of SIM-loaded PRF as an effective, biocompatible, and patient-friendly approach to enhance bone regeneration and implant success. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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13 pages, 3389 KB

Open AccessArticle

Kinetics of MM1.S Multiple Myeloma Cells in a 3D Polymer Particle Culture System with Bone Marrow Stromal Cells and Bortezomib

by Shin Aizawa, Miyuki Yuda, Shuichi Hirai, Isao Tsuboi, Takashi Koike, Yoshihiro Hatta, Katsuhiro Miura and Masahiro Yasuda

Pharmaceuticals 2026, 19(1), 122; https://doi.org/10.3390/ph19010122 - 10 Jan 2026

Viewed by 562

Abstract

Background: Three-dimensional (3D) culture systems use polymer particles with a bone marrow stroma cell feeder layer to reproduce a biostructural hematopoiesis state more effectively than in conventional two-dimensional (2D) culture methods. The 3D culture maintains normal hematopoiesis, resulting in prolongation of hematopoietic stem cell proliferation and differentiation, while the bone marrow stromal cells in the culture alter the growth of leukemic cells and protect them from anticancer agents. However, the effect of stromal cells on hematopoietic stem cell proliferation and differentiation and neoplastic cells, including leukemia, in 3D culture is still a point of contention. Methods: We assessed the mechanism of two different bone-marrow-derived stromal cells (i.e., MS-5 and Tst-4) with different characteristics by using a feeder layer in the 3D culture to compare their supportive action on leukemic cells, focusing on the role of 3D cultures constructed with bone marrow stromal cells in leukemic cell growth. Multiple myeloma cells are strongly related to stromal cells in their proliferation; hence, cloned MM1.S cells derived from multiple myeloma were cocultured in 3D, and their cell growth was examined. We also examined the effect of the antineoplastic agent bortezomib, a proteasome inhibitor, in the 3D culture system with a different stromal cell feeder. Results and Conclusions: When MM1.S myeloma cells were cultured with MS-5 stroma in 3D conditions, cell growth was found to be slow compared with that in 2D culture, as well as with those in both the 2D and 3D cocultures with Tst-4 stroma. Additionally, the MS-5 cells in the 3D culture protected the MM1.S cells from the cytocidal effect of the bortezomib treatment. Different MM1.S cell kinetics were observed depending on the stromal cells used, suggesting their inherent and complicated characteristics. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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16 pages, 2599 KB

Open AccessArticle

GLUT1-DS Brain Organoids Exhibit Increased Sensitivity to Metabolic and Pharmacological Induction of Epileptiform Activity

by Loïc Lengacher, Sylvain Lengacher, Pierre J. Magistretti and Charles Finsterwald

Pharmaceuticals 2026, 19(1), 105; https://doi.org/10.3390/ph19010105 - 7 Jan 2026

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Abstract

Background/Objectives: Glucose Transporter 1 Deficiency Syndrome (GLUT1-DS) is a neurodevelopmental disorder caused by mutations in the gene encoding glucose transporter 1 (GLUT1), which leads to impaired glucose transport into the brain and is characterized by drug-resistant epilepsy. Limited glucose supply disrupts neuronal and astrocytic energy homeostasis, but how hypometabolism translates into network hyperexcitability remains poorly understood. Here, we used induced pluripotent stem cells (iPSCs)-derived brain organoids to examine how reduced metabolic substrate availability shapes epileptiform dynamics in human neuronal circuits from GLUT1-DS. Methods: Brain organoids were generated from a healthy donor or a GLUT1-DS patient and interfaced with multielectrode arrays (MEA) for recording of neuronal activity. A unified Python (v3.10)-based analytical pipeline was developed to quantify spikes, bursts, and power spectral density (PSD) across frequency bands of neuronal activity. Organoids were challenged with reduced glucose, pentylenetetrazol (PTZ), potassium chloride (KCl), and tetrodotoxin (TTX) to assess metabolic and pharmacological modulation of excitability. Results: GLUT1-DS organoids exhibited elevated baseline hyperexcitability compared to healthy control, characterized by increased spike rates, prolonged bursts, increased spikes per burst, and elevated PSD. Reduced glucose availability further amplified these features selectively in GLUT1-DS. Conclusions: Human brain organoids reproduce the pathological coupling between hypometabolism and hyperexcitability in GLUT1-DS. Our platform provides a mechanistic model and quantification tool for evaluating metabolic and anti-epileptic therapeutic strategies. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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18 pages, 5900 KB

Open AccessArticle

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Modulate Chemoradiotherapy Response in Cervical Cancer Spheroids

by Kesara Nittayaboon, Piyatida Molika, Rassanee Bissanum, Kittinun Leetanaporn, Nipha Chumsuwan and Raphatphorn Navakanitworakul

Pharmaceuticals 2025, 18(7), 1050; https://doi.org/10.3390/ph18071050 - 17 Jul 2025

Cited by 3 | Viewed by 1543

Abstract

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are significant in chemo- and radiotherapy resistance. Previous research has focused on BM-MSCs, demonstrating their functional involvement in cancer progression as mediators in the tumor microenvironment. They play multiple roles in tumorigenesis, angiogenesis, and metastasis. BM-MSC-derived exosomes (BM-MSCs-exo) are small vesicles, typically 50–300 nm in diameter, isolated from BM-MSCs. Some studies have demonstrated the tumor-suppressive effects of BM-MSCs-exo. Objective: This study aimed to investigate their role in modulating the impact of chemoradiotherapy (CRT) in different types of cervical cancer spheroid cells. Methods: The spheroids after treatment were subject to size measurement, cell viability, and caspase activity. Then, the molecular mechanism was elucidated by Western blot analysis. Results: We observed a reduction in spheroid size and an increase in cell death in HeLa spheroids, while no significant changes in size or cell viability were found in SiHa spheroids. At the molecular level, CRT treatment combined with BM-MSCs-exo in HeLa spheroids induced apoptosis through the activation of the NF-κB pathway, specifically via the NF-κB1 (P50) transcription factor, leading to the upregulation of apoptosis-related molecules. In contrast, CRT combined with BM-MSCs-exo in SiHa spheroids exhibited an opposing effect: although cellular viability decreased, caspase activity also decreased, which correlated with increased HSP27 expression and the subsequent downregulation of apoptotic molecules. Conclusion: Our study provides deeper insight into the potential of BM-MSCs-exo in cervical cancer treatment, supporting the development of more effective and safer therapeutic strategies for clinical application. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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21 pages, 73459 KB

Open AccessArticle

Impact of Post-Thaw Enrichment of Primary Human Hepatocytes on Steatosis, Inflammation, and Fibrosis in the TruVivo^® System

by Justin J. Odanga, Sharon M. Anderson, Sharon C. Presnell, Edward L. LeCluyse, Jingsong Chen and Jessica R. Weaver

Pharmaceuticals 2024, 17(12), 1624; https://doi.org/10.3390/ph17121624 - 3 Dec 2024

Cited by 1 | Viewed by 2093

Abstract

Background: Liver diseases are a global health concern. Many in vitro liver models utilize cryopreserved primary human hepatocytes (PHHs), which commonly undergo post-thaw processing through colloidal silica gradients to remove debris and enrich for a viable PHH population. Post-thaw processing effects on healthy PHHs are partially understood, but the consequences of applying disease-origin PHHs to post-thaw density gradient separation have not been described. Methods: Using the TruVivo^® system, diseased, type 2 diabetes mellitus (T2DM), and fibrotic PHHs were cultured for 14 days after initially being subjected to either low-density (permissive) or high-density (selective) gradients using Percoll-based thawing medium. Results: Changes in functionality, including albumin and urea secretion and CYP3A4 activity, were measured in diseased, T2DM, and fibrotic PHHs enriched in low Percoll compared to PHHs enriched in high Percoll. Lipogenesis increased in the PHHs enriched in low Percoll. Higher expression of CK18 and TGF-β, two fibrotic markers, and changes in expression of the macrophage markers CD68 and CD163 were also measured. Conclusions: The use of Percoll for the enrichment of PHHs post-thaw results in differences in attachment and functionality, along with changes in diseased phenotypes, in the TruVivo^® system. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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11 pages, 4429 KB

Open AccessArticle

Comparison of Glioblastoma Cell Culture Platforms Based on Transcriptional Similarity with Paired Tissue

by Junseong Park, Ilkyoo Koh, Junghwa Cha, Yoojung Oh, Jin-Kyoung Shim, Hyejin Kim, Ju Hyung Moon, Eui Hyun Kim, Jong Hee Chang, Pilnam Kim and Seok-Gu Kang

Pharmaceuticals 2024, 17(4), 529; https://doi.org/10.3390/ph17040529 - 19 Apr 2024

Cited by 5 | Viewed by 3369

Abstract

No standardized in vitro cell culture models for glioblastoma (GBM) have yet been established, excluding the traditional two-dimensional culture. GBM tumorspheres (TSs) have been highlighted as a good model platform for testing drug effects and characterizing specific features of GBM, but a detailed evaluation of their suitability and comparative performance is lacking. Here, we isolated GBM TSs and extracellular matrices (ECM) from tissues obtained from newly diagnosed IDH1 wild-type GBM patients and cultured GBM TSs on five different culture platforms: (1) ordinary TS culture liquid media (LM), (2) collagen-based three-dimensional (3D) matrix, (3) patient typical ECM-based 3D matrix, (4) patient tumor ECM-based 3D matrix, and (5) mouse brain. For evaluation, we obtained transcriptome data from all cultured GBM TSs using microarrays. The LM platform exhibited the most similar transcriptional program to paired tissues based on GBM genes, stemness- and invasiveness-related genes, transcription factor activity, and canonical signaling pathways. GBM TSs can be cultured via an easy-to-handle and cost- and time-efficient LM platform while preserving the transcriptional program of the originating tissues without supplementing the ECM or embedding it into the mouse brain. In addition to applications in basic cancer research, GBM TSs cultured in LM may also serve as patient avatars in drug screening and pre-clinical evaluation of targeted therapy and as standardized and clinically relevant models for precision medicine. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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Review

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24 pages, 1076 KB

Open AccessReview

Bioengineering Stem Cell-Derived Glioblastoma Organoids: A Comprehensive Review

by Alexandra D. Avera and Yonghyun Kim

Pharmaceuticals 2025, 18(12), 1830; https://doi.org/10.3390/ph18121830 - 1 Dec 2025

Viewed by 1491

Abstract

The development of novel therapeutics for deadly diseases such as glioblastoma (GBM) is bottlenecked by poor preclinical models. GBM is the most common and deadliest primary brain tumor in adults, with an average prognosis of 12–15 months, primarily due to its high cellular heterogeneity and treatment resistance from GBM stem cells. The advancement of in vitro models into organoids, three-dimensional tissue-like modeling systems, has been a promising approach to improving translational medicine for GBM. However, the critical tradeoff between technical convenience and physiological relevance threatens the integrity and reproducibility of GBM organoid (GBO) biomanufacturing. This comprehensive review breaks down and discusses the key features of GBM tumor microenvironment (TME), traces the advancement of in vitro models from two-dimensional cultures to three-dimensional stem cell-derived GBOs, evaluates the process through an engineering perspective (genetic, biochemical, biophysical, and process engineering), and addresses critical translational gaps. Reviewing trends over the last fifteen years in biomanufacturing approaches to GBOs revealed fundamental oversights that address previous review focuses on the limitations of organoids (i.e., maturity, vasculature, and immune defense). To summarize, GBO’s translational gap and reproducibility challenges are rooted in the prioritization of technical convenience over physiological relevance. To achieve clinical relevance, future GBO development must focus on transitioning to fully defined components (excluding animal-derived ECM), developing sufficiently large-sized constructs to recapitulate the full TME, and integrating non-destructive and enhanced functional readouts of the GBOs. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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24 pages, 1486 KB

Open AccessReview

Next-Generation Hydrogels for Biliary Organoid Engineering

by Andrea Marfoglia and Giovanni Sorrentino

Pharmaceuticals 2025, 18(12), 1781; https://doi.org/10.3390/ph18121781 - 23 Nov 2025

Viewed by 1796

Abstract

The biliary tree is a fundamental structural and functional component of the liver, lined with cholangiocytes which control bile flow and regulate bile homeostasis. In addition to their physiological roles, cholangiocytes are involved in pathological processes known as cholangiopathies. These biliary disorders significantly impair liver function, and their effects are often irreversible, making liver transplantation the only curative option. This substantial clinical burden highlights the need for innovative bioengineered strategies to study disease mechanisms and to restore or replace biliary tissue. In this framework, biliary organoids offer a robust platform to model liver diseases in vitro with physiological accuracy. Compared with traditional 2D or explant-based systems, organoids provide higher physiological relevance, patient specificity, and scalability, although challenges remain in standardization and clinical translation. Organoids are traditionally cultured within basement membrane extract (BME) matrices, which are commercially available under various names. While BME-based matrices support organoid growth and function, their undefined composition, variability, and animal origin limit reproducibility and clinical translation. These drawbacks have driven the development of alternative matrices based on engineered hydrogels. Hydrogels, whether of natural or synthetic origin, provide chemically defined and tunable environments that allow independent modulation of their biochemical and biophysical properties. Acting at the interface between materials science and biology, they enable the creation of microenvironments with precisely controlled cues. In this review, we summarize advances in biliary organoid bioengineering and discuss how hydrogel-based systems are shaping next-generation platforms for organoid growth, differentiation, and disease modeling toward more translationally relevant biliary models. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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27 pages, 1853 KB

Open AccessReview

Two- and Three-Dimensional Culture Systems: Respiratory In Vitro Tissue Models for Chemical Screening and Risk-Based Decision Making

by Joanne Wallace, Mary C. McElroy, Mitchell Klausner, Richard Corley and Seyoum Ayehunie

Pharmaceuticals 2025, 18(1), 113; https://doi.org/10.3390/ph18010113 - 16 Jan 2025

Cited by 8 | Viewed by 4437

Abstract

Risk of lung damage from inhaled chemicals or substances has long been assessed using animal models. However, New Approach Methodologies (NAMs) that replace, reduce, and/or refine the use of animals in safety testing such as 2D and 3D cultures are increasingly being used to understand human-relevant toxicity responses and for the assessment of hazard identification. Here we review 2D and 3D lung models in terms of their application for inhalation toxicity assessment. We highlight a key case study for the Organization for Economic Cooperation and Development (OECD), in which a 3D model was used to assess human toxicity and replace the requirement for a 90-day inhalation toxicity study in rats. Finally, we consider the regulatory guidelines for the application of NAMs and potential use of different lung models for aerosol toxicity studies depending on the regulatory requirement/context of use. Full article

(This article belongs to the Special Issue 2D and 3D Culture Systems: Current Trends and Biomedical Applications)

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