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Biomedicines
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Innovative Approaches in In Vitro Models: From Design to Application
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Innovative Approaches in In Vitro Models: From Design to Application

A special issue of Biomedicines (ISSN 2227-9059). This special issue belongs to the section "Biomedical Engineering and Materials".

Deadline for manuscript submissions: 31 July 2026 | Viewed by 2086

Editor

Dr. Tina Maver

E-Mail Website
Guest Editor
1. The Institute of Biomedical Sciences, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia
2. The Department of Pharmacology, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia
Interests: polysaccharide-based hydrogels; 3D bioprinting and skin-on-a-chip models; wound dressings; controlled drug delivery; regenerative and in vitro tissue models
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue, titled “Innovative Approaches in In Vitro Models: From Design to Application”, will focus on the development and application of advanced in vitro systems that more accurately mimic human physiology and pathology. These models are essential for understanding disease mechanisms, drug responses, and regenerative processes, while reducing reliance on animal testing. This issue welcomes contributions covering novel cell isolation and characterization techniques that enable the use of primary human cells and patient-derived tissues for personalized model design. Emphasis will be placed on innovative model platforms such as 3D-bioprinted tissues, organ-on-a-chip systems, microfluidic devices, and multi-layer constructs that reproduce key aspects of human organs and diseases, including vascularization, inflammation, fibrosis, and wound healing. Technological advances in biosensing, imaging, and microfabrication, which support dynamic and functional readouts, are also of great interest. By bridging cell biology, bioengineering, and translational medicine, this Special Issue aims to highlight integrative approaches that enhance the physiological relevance, predictive power, and clinical applicability of next-generation in vitro models for studying human disease.

Dr. Tina Maver
Guest Editor

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Keywords

  • in vitro disease models
  • human cell isolation and characterization
  • organ-on-a-chip and microfluidic systems
  • 3D bioprinting and tissue engineering
  • translational biomedical research

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Published Papers (4 papers)

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26 pages, 4505 KB  
Article
Functional In Vitro Model of the Canine Corpus Luteum: Isolation, Culture and Characterization of Steroidogenically Active Luteal Cells
by Patrycja Kalak, Paulina Bugno, Jan P. Madej, Mateusz Speruda, Antoni Szumny, Maciej Janeczek, Wojciech Niżański, Tomasz Gębarowski and Michał Dzięcioł
Biomedicines 2026, 14(7), 1444; https://doi.org/10.3390/biomedicines14071444 (registering DOI) - 25 Jun 2026
Abstract
Background/Objectives: The corpus luteum (CL) in the dog is the sole source of progesterone (P4) during diestrus and pregnancy, making it a key regulator of reproductive function. However, robust and functionally validated in vitro models of canine luteal cells remain limited. This study [...] Read more.
Background/Objectives: The corpus luteum (CL) in the dog is the sole source of progesterone (P4) during diestrus and pregnancy, making it a key regulator of reproductive function. However, robust and functionally validated in vitro models of canine luteal cells remain limited. This study aimed to establish and characterize a reproducible primary culture system of canine luteal cells with preserved steroidogenic activity and regulatory responsiveness. Methods: Ovaries containing CLs were collected from five clinically healthy bitches undergoing routine ovariohysterectomy (OHE). Luteal tissue was mechanically fragmented and enzymatically digested using collagenase type II. Primary cultures were established using an explant-based approach and maintained in Dulbecco’s Modified Eagle Medium/Ham’s F-12 nutrient mixture (DMEM/F12) or Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 20% fetal bovine serum (FBS). Cellular morphology, proliferation, expression of steroidogenic markers—steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), P4 secretion, and responsiveness to forskolin stimulation were evaluated. Results: Cultured luteal cells exhibited stable attachment, proliferation, and a predominantly spindle-shaped morphology. Both media supported maintenance of a steroidogenic phenotype, while RPMI 1640 enabled enhanced proliferation, allowing expansion up to passage three and efficient cryobanking. Cells remained functionally active, secreting progesterone for up to 28 days in vitro. Forskolin stimulation increased progesterone secretion up to 2.7-fold, confirming preserved cyclic AMP-dependent steroidogenic responsiveness. Conclusions: The canine CL is a reliable source of functionally competent luteal cells, and the established culture system represents a physiologically relevant in vitro model. To our knowledge, this is the first functionally validated in vitro model of the canine CL. This platform enables controlled investigations of luteal function, endocrine regulation, and mechanisms of P4 synthesis, supporting its application in mechanistic and translational reproductive research. Full article
(This article belongs to the Special Issue Innovative Approaches in In Vitro Models: From Design to Application)
16 pages, 5635 KB  
Article
Accurate Quantification of Platelets and Leukocytes in Platelet-Rich Fibrin Using Direct Fibrinolytic Digestion: Effects of Centrifugation Protocols on Cellular Composition and Fibrin Architecture
by Pattheera Apaiso, Wutigri Nimlamool, Teerada Daroontum and Supatra Sangin
Biomedicines 2026, 14(5), 1096; https://doi.org/10.3390/biomedicines14051096 - 13 May 2026
Viewed by 482
Abstract
Background/Objectives: Platelet-rich fibrin (PRF) is widely used in regenerative medicine and dentistry because of its ability to deliver growth factors and cellular components that support tissue healing. However, accurate quantification of platelets and leukocytes within PRF matrices remains challenging, as indirect subtraction-based [...] Read more.
Background/Objectives: Platelet-rich fibrin (PRF) is widely used in regenerative medicine and dentistry because of its ability to deliver growth factors and cellular components that support tissue healing. However, accurate quantification of platelets and leukocytes within PRF matrices remains challenging, as indirect subtraction-based methods may not reliably reflect the true cellular composition. This study aimed to apply a direct fibrinolytic digestion–flow cytometry approach for precise cellular quantification and to systematically evaluate the effects of different centrifugation protocols on PRF cellular composition and fibrin architecture. Methods: PRF clots were generated using high-speed protocols (leukocyte- and platelet-rich fibrin [L-PRF] and concentrated growth factors [CGF]) and low-speed protocols (advanced PRF [A-PRF] and A-PRF+). Cellular content was quantified using a fibrinolytic digestion-based approach with tissue plasminogen activator, followed by flow cytometry analysis. Histological evaluation was performed to assess fibrin architecture and cellular distribution. Results: L-PRF and CGF exhibited significantly greater platelet enrichment than A-PRF. T-lymphocyte counts were elevated across all PRF groups, with significantly higher levels observed in L-PRF and CGF than in A-PRF. Monocyte and neutrophil levels were reduced following PRF preparation; however, A-PRF retained significantly higher neutrophil levels than CGF, whereas B-lymphocyte levels showed a moderate increase without significant intergroup differences. Histologically, high-speed protocols produced pronounced cellular stratification and denser fibrin networks, whereas low-speed protocols demonstrated a more homogeneous leukocyte distribution within a porous fibrin matrix. Conclusions: Centrifugation protocols significantly influence PRF cellular composition and fibrin architecture. The direct fibrinolytic digestion–flow cytometry approach provides a reliable method for accurate cellular quantification and may facilitate the standardization and optimization of PRF preparation protocols for regenerative applications. Full article
(This article belongs to the Special Issue Innovative Approaches in In Vitro Models: From Design to Application)
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16 pages, 2733 KB  
Article
Engineering Bone-Mimetic Microspheres to Recapitulate the Tumor Microenvironment for In Vitro Osteosarcoma Modeling
by Fangqiao Zheng, Zhengyi Lan, Hangrong Chen and Ming Ma
Biomedicines 2026, 14(4), 868; https://doi.org/10.3390/biomedicines14040868 - 10 Apr 2026
Viewed by 743
Abstract
Background: Osteosarcoma (OS) is an aggressive bone tumor. The lack of physiologically relevant three-dimensional models that recapitulate the native tumor microenvironment hampers drug development and mechanistic studies. The study aimed to develop bone-mimetic microspheres for the construction of an OS model. Materials and [...] Read more.
Background: Osteosarcoma (OS) is an aggressive bone tumor. The lack of physiologically relevant three-dimensional models that recapitulate the native tumor microenvironment hampers drug development and mechanistic studies. The study aimed to develop bone-mimetic microspheres for the construction of an OS model. Materials and Methods: We employed droplet microfluidics to fabricate bone-mimetic microspheres (named MSHA) from a composite of gelatin methacryloyl, polyethylene glycol diacrylate, and nano-hydroxyapatite (nHA). MNNG/HOS cells were cultured on MSHA microspheres and subsequently evaluated for their bioactivity and capabilities of stemness, migration, and invasion. Results: The microfluidic platform enabled efficient and scalable production of highly uniform MSHA microspheres with controlled sizes. MNNG/HOS cells cultured on MSHA maintained high viability and spontaneously formed compact tumor spheroids after 7 days. Compared with two-dimensional cultures, cells cultured on these microsphere-based platforms exhibited enhanced migration and invasion capacities, along with increased expression of relevant biomarkers. RNA sequencing further revealed the activation of cancer-related pathways. Notably, the incorporation of nHA into microspheres amplified these malignant phenotypes, potentially through the activation of ECM–receptor interaction and calcium signaling pathways. Conclusions: The microfluidics-fabricated MSHA microspheres, as biomimetic three-dimensional culture scaffolds, offer a promising platform for applications in mechanistic studies of osteosarcoma progression and drug screening. Full article
(This article belongs to the Special Issue Innovative Approaches in In Vitro Models: From Design to Application)
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11 pages, 814 KB  
Brief Report
Modeling Blood–Brain Barrier Efflux Transport Using a Breast Cancer Resistance Protein Overexpression Cell Line
by Alexandra E. Meyer, Natalie G. Alexander, Elisa M. Tucker, Hallie E. Knight, Benjamin T. Klemp, Bryan J. Estrada, Sarah F. Hathcock, Henry D. Mauser, Kylie A. Buchanan and Brandon J. Kim
Biomedicines 2026, 14(6), 1192; https://doi.org/10.3390/biomedicines14061192 - 25 May 2026
Viewed by 468
Abstract
Background: The blood–brain barrier (BBB) separates the circulation from the central nervous system (CNS) and serves to maintain brain homeostasis. The BBB comprises highly specialized brain endothelial cells (BECs) with unique properties that allow the BBB to maintain strict regulation of molecules [...] Read more.
Background: The blood–brain barrier (BBB) separates the circulation from the central nervous system (CNS) and serves to maintain brain homeostasis. The BBB comprises highly specialized brain endothelial cells (BECs) with unique properties that allow the BBB to maintain strict regulation of molecules entering and exiting the CNS. These characteristics include tight junctions, low endocytosis rates, and efflux and nutrient transporters. Breast cancer resistance protein (BCRP) is an efflux transporter found at the BBB that plays a key role in protecting the CNS. Together with other efflux transporters, BCRP contributes to multidrug-resistant cancers and difficulty delivering drugs and therapeutics to the brain and other organs. Methods: Using the hCMEC/D3 line, we utilized BCRP substrate rosuvastatin to effectively select for cells expressing high amounts of BCRP, thus generating hCMEC/D3-BCRP. To assess protein abundance, we utilized flow cytometry and confirmed expression via qPCR. To investigate BCRP efflux function in evolved hCMEC/D3-BCRP, we performed substrate accumulation assays with BCRP and P-gp substrates. Results: We found hCMEC/D3-BCRP had increased BCRP abundance and expression relative to parent hCMEC/D3. We also observed an increase in BCRP function via substrate accumulation of two BCRP substrates compared to parent hCMEC/D3. Conclusions: BCRP serves a protective role within the BBB and is a major hurdle in drug delivery. We generated a BCRP overexpression BEC cell line (hCMEC/D3-BCRP) under the influence of endogenous promoters. This cell line can be used to further investigate the role of BCRP in BECs and utilized in efflux transport studies. Full article
(This article belongs to the Special Issue Innovative Approaches in In Vitro Models: From Design to Application)
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