Liver Microarchitecture-Guided Design of Biomimetic Scaffolds: Biomaterials, Dynamic Culture Systems, and Emerging Clinical Translation: A Review.

The traditional two-dimensional (2D) cell culture methods have a long history of mimicking in vivo cell growth. However, these methods cannot fully represent physiological conditions, which lack two major indexes of the in vivo environment; one is a three-dimensional 3D cell environment, and the other is mechanical stimulation; therefore, they are incapable of replicating the essential cellular communications between cell to cell, cell to the extracellular matrix, and cellular responses to dynamic mechanical stimulation in a physiological condition of body movement and blood flow. To solve these problems and challenges, 3D cell carriers have been gradually developed to provide a 3D matrix-like structure for cell attachment, proliferation, differentiation, and communication in static and dynamic culture conditions. 3D cell carriers in dynamic culture systems could primarily provide different mechanical stimulations which further mimic the real in vivo microenvironment. In this review, the current advances in 3D dynamic cell culture approaches have been introduced, with their advantages and disadvantages being discussed in comparison to traditional 2D cell culture in static conditions.

Cells can sense and respond to different kinds of continuous mechanical strain in the human body. Mechanical stimulation needs to be included within the in vitro culture system to better mimic the existing complexity of in vivo biological systems. Existing commercial dynamic culture systems are generally two-dimensional (2D) which fail to mimic the three-dimensional (3D) native microenvironment. In this study, a pneumatically driven fiber robot has been developed as a platform for 3D dynamic cell culture. The fiber robot can generate tunable contractions upon stimulation. The surface of the fiber robot is formed by a braiding structure, which provides promising surface contact and adequate space for cell culture. An in-house dynamic stimulation using the fiber robot was set up to maintain NIH3T3 cells in a controlled environment. The biocompatibility of the developed dynamic culture systems was analyzed using LIVE/DEAD™ and alamarBlue™ assays. The results showed that the dynamic culture system was able to support cell proliferation with minimal cytotoxicity similar to static cultures. However, we observed a decrease in cell viability in the case of a high strain rate in dynamic cultures. Differences in cell arrangement and proliferation were observed between braided sleeves made of different materials (nylon and ultra-high molecular weight polyethylene). In summary, a simple and cost-effective 3D dynamic culture system has been proposed, which can be easily implemented to study complex biological phenomena in vitro.

Static and dynamic culture of human endothelial cells encapsulated inside alginate-gelatin microspheres

Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems

Testicular tissue engineering for in vitro spermatogenesis aims to restore fertility, focusing on challenges like efficiency, ethical concerns, and the need for a deeper biological understanding. The use of decellularized scaffolds led to better cell seeding and differentiation, and exosomes led to enhanced spermatogenesis. Also, the dynamic culture systems are being explored to replicate in vivo conditions more accurately. In this study, we aimed to utilize a perfusion mini-bioreactor for the dynamic culture of mouse spermatogonial stem cells on decellularized testicular matrix plates supplemented with exosomes. Our goal was to assess the progression of the spermatogenesis process through histological, immunohistochemical, and molecular analyses over four weeks. Human testicular tissues were decellularized using 1% sodium dodecyl sulfate and were then fabricated into thin plates using a cryostat. Sertoli and spermatogonial stem cells were isolated from neonate mouse testis and seeded onto the decellularized testicular matrix plates. A mini-perfusion bioreactor was employed to create dynamic culture conditions. Also, MSCs-derived exosomes were introduced to the culture medium, alone or in combination with a spermatogenic medium containing numerous chemical factors. The histological, IHC, and molecular analyses were performed at the end of the experiment. Our decellularization procedure successfully preserved the ECM components, while eliminating native cells. The isolated cells expressed PLZF and VIMENTIN markers, confirming the presence of SSCs and Sertoli cells. The seeded scaffolds exhibited proper homing, viability, proliferation, and differentiation of the cells towards in vitro spermatogenesis. Also, exosome treatment is capable of enhancing the spermatogenic potential of SSCs. Our findings indicate that the dynamic culture system significantly promoted the proliferation and differentiation of SSCs into mature spermatozoa. The use of exosomes further enhanced these effects, as evidenced by improved cellular viability, reduced apoptosis, and advanced spermatogenesis to the elongated spermatid stage. The combined treatment of exosomes and spermatogenic medium showed a synergistic effect, yielding superior outcomes in terms of sperm cell maturity and functionality. This study underscores the potential of combining decellularized testicular matrices with exosome therapy in a dynamic culture set up to advance the field of reproductive biology and fertility restoration.

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.

Study question Is improvement of follicle growth and health during dynamic culture in Perifusion Bioreactor (PB) associated to a healthier condition and remodeling of extracellular matrix? Summary answer The dynamic culture in PB allows the extracellular matrix (ECM) network rearrangement improving follicle growth and viability compared to static culture What is known already Over the past two decades, in vitro folliculogenesis has achieved moderate success in large mammals, emerging as a distinctive and reliable method for obtaining secondary follicles. Recently, although dynamic culture in bioreactors has been demonstrated to enhance follicle progression, quality, and viability compared to static culture, less emphasis has been devoted to ECM remodeling. Ovarian tissue ECM plays a crucial role in cell–cell, and cell-ECM interactions involved in follicle activation and growth. The comprehension of ECM function deepens our understanding of follicular development and holds significant promise for future reproductive technologies Study design, size, duration Bovine ovaries (age 8-24 months) were collected at slaughterhouse. BOCT strips (1x1x0.5mm) from the same ovary were cultured in groups of 10 for 14 days in PB (dynamic culture) and conventional dishes (CD, static culture). At the end of culture, collagen thickness and packing density was assessed through PicroSirius red (PSR) staining, follicle stages and quality through histology, and follicle viability through live-dead far-red and propidium iodide labeling under confocal microscopy Participants/materials, setting, methods Neosynthesis and remodeling of collagen fibers was analyzed on PSR-stained samples under polarized light measuring the color of each pixel. A color threshold was set to isolate the four colors seen in PSR-stained samples under polarized light: green, thin fibers (neosynthesized); yellow, mid-sized fibers (low assembly); orange, mid-sized fibers (medium assembly); red, mature thick fibers (high assembly). The color threshold was set as follows: red 2–9, yellow 39-51, orange 10–38, green 52–128 Main results and the role of chance In fresh tissue, analysis of PSR-stained samples showed the following values: Red 1.35; Orange 4.93; Yellow 0.18; Green 0.15. At Day 14, compared to the static culture, culture in PB, exhibits a significantly higher production of green (PB 0.22 vs CD 0.02 P < 0.01) and yellow (PB 0.56 vs CD 0.26 P < 0.05) fibers indicating an improved neosynthesis and remodeling of collagen and ECM health condition. Such higher ECM remodeling after dynamic culture is associated to a significantly higher percentage of secondary follicles (PB 15.4– CD 4.1%, P < 0.01 ), superior follicle quality (grade I/II, PB 70,5 - CD 28.4%, P < 0.01) and viability (PB 70 – CD 41.1%, P < 0.01). PSR analysis suggests that the continuous nutrients and oxygen supply, catabolites removal, and biomechanical stimulation during dynamic PB culture, stimulates the deposition of newly synthesized collagen which in turn, promotes follicle growth Limitations, reasons for caution Although the bovine is a suitable reproductive model for human, such findings should be replicated on human ovarian tissue Wider implications of the findings The aim of ovarian tissue culture is to promote the growth of healthy secondary follicles that can be isolated for further culture to generate MII oocytes. The present findings suggest that adoption of dynamic ovarian tissue culture could enhance the outcome of folliculogenesis in vitro Trial registration number not applicable

The culture environment plays an important role for stem cells’ cultivation. Static or dynamic culture preserve differential potentials to affect human mesenchymal stem cells’ (hMSCs) proliferation and differentiation. In this study, hMSCs were seeded on fiber disks and cultured in a bidirectional-flow bioreactor or spinner-flask bioreactor with a supplement of osteogenic medium. The hMSCs’ proliferation, osteogenic differentiation, and extracellular matrix deposition of mineralization were demonstrated. The results showed that the spinner flask improved cell viability at the first two weeks while the bidirectional-flow reactor increased the cell proliferation of hMSCs through the four-week culture period. Despite the flow reactor having a higher cell number, a lower lactose/glucose ratio was noted, revealing that the bidirectional-flow bioreactor provides better oxygen accessibility to the cultured cells/disk construct. The changes of calcium ions in the medium, the depositions of Ca2+ in the cells/disk constructs, and alkaline phosphate/osteocalcin activities showed the static culture of hMSCs caused cells to mineralize faster than the other two bioreactors but without cell proliferation. Otherwise, cells were distributed uniformly with abundant extracellular matrix productions using the flow reactor. This reveals that the static and dynamic cultivations regulated the osteogenic process differently in hMSCs. The bidirectional-flow bioreactor can be used in the mass production and cultivation of hMSCs for applications in bone regenerative medicine.

Sequential construction of vascularized and mineralized bone organoids using engineered ECM-DNA-CPO-based bionic matrix for efficient bone regeneration.

Microfluidic dynamic embryo culture increases the production of top quality human embryos through reduction in embryo fragmentation

Presently, the majority of cells are cultivated by two-dimensional (2D) methods; however, latest and enhanced procedures employing three-dimensional (3D) cell culturing techniques provide strong indications that significantly more sophisticated studies can be carried out, providing invaluable insights. Recent years have seen a rapid development of 3D cell cultures since cells grown in a static environment on a flat substrate are far from reaching an in vivo condition. Currently, scientists are gradually realising that in vitro cell shape, structure, and physiological activities may be achieved. As a resolution, a three-dimensional matrix-like framework for cell attachment, proliferation, differentiation, and communication in both static and dynamic culture conditions is what three-dimensional cell carriers have gradually come to offer. Different mechanical stimulations that more closely resemble the genuine in vivo microenvironment could be the main function of 3D cell carriers in dynamic culture systems. Current developments in 3D dynamic cell culture techniques have been presented in this review, along with a discussion of their benefits and drawbacks when compared to conventional 2D cell growth under static settings.

Enmotion (embryo’s natural motion): a paired randomized controlled trial demonstrating pregnancy rates are equivalent between static and dynamic culture systems

Polydimethylsiloxane (PDMS) is a silicone-based synthetic material used in various biomedical applications due to its properties, including transparency, flexibility, permeability to gases, and ease of use. Though PDMS facilitates and assists the fabrication of complicated geometries at micro- and nano-scales, it does not optimally interact with cells for adherence and proliferation. Various strategies have been proposed to render PDMS to enhance cell attachment. The majority of these surface modification techniques have been offered for a static cell culture system. However, dynamic cell culture systems such as organ-on-a-chip devices are demanding platforms that recapitulate a living tissue microenvironment’s complexity. In organ-on-a-chip platforms, PDMS surfaces are usually coated by extracellular matrix (ECM) proteins, which occur as a result of a physical and weak bonding between PDMS and ECM proteins, and this binding can be degraded when it is exposed to shear stresses. This work reports static and dynamic coating methods to covalently bind collagen within a PDMS-based microfluidic device using polydopamine (PDA). These coating methods were evaluated using water contact angle measurement and atomic force microscopy (AFM) to optimize coating conditions. The biocompatibility of collagen-coated PDMS devices was assessed by culturing primary human bronchial epithelial cells (HBECs) in microfluidic devices. It was shown that both PDA coating methods could be used to bind collagen, thereby improving cell adhesion (approximately three times higher) without showing any discernible difference in cell attachment between these two methods. These results suggested that such a surface modification can help coat extracellular matrix protein onto PDMS-based microfluidic devices.

End-stage hepatic failure is a potentially life-threatening condition for which orthotopic liver transplantation (OLT) is the only effective treatment. However, a shortage of available donor organs for transplantation each year results in the death of many patients waiting for liver transplantation. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation. However, primary hepatocyte transplantation has rarely produced therapeutic effects because mature hepatocytes cannot be effectively expanded in vitro, and the availability of hepatocytes is often limited by shortages of donor organs. Decellularization is an attractive technique for scaffold preparation in stem cell-based liver engineering, as the resulting material can potentially retain the liver architecture, native vessel network and specific extracellular matrix (ECM). Thus, the reconstruction of functional and practical liver tissue using decellularized scaffolds becomes possible. This review focuses on the current understanding of liver tissue engineering, whole-organ liver decellularization techniques, cell sources for recellularization and potential clinical applications and challenges.

We have previously demonstrated that purmorphamine exerts an osteoinductive effect on human bone marrow mesenchymal stem cells (hBM-MSCs) in a two-dimensional (2D) static culture system. In the present study, we have evaluated the effect of purmorphamine on osteogenic differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) dynamic culture system vs. a 2D static culture condition. In this study, hMSCs were seeded in either 3D collagen scaffolds using a perfusion bioreactor or on 2D culture plates. One day later, the medium was replaced with osteogenic medium supplemented with dexamethasone, l‐ascorbic acid and β-glycerophosphate in the presence or absence of purmorphamine. Histological staining, flow cytometry, and real-time PCR were conducted for evaluation of osteogenesis in the study groups. The expressions of RUNX-2, alkaline phosphatase (ALP), osteocalcin (OC), collagen I (Col I), and bone sialoprotein (BSP) were compared between the 3D and 2D culture systems at 14 and 21 days post-induction of differentiation. Based on our results, alizarin red staining showed deposition of the mineralized matrix in the 2D static and 3D dynamic cultures. Our flow cytometric analysis indicated that the number of ALP+/OC− cells increased in the presence of purmorphamine in the 2D culture rather than the 3D system at day14. One week later, the numbers of OC+/ALP− and OC+/Stro-1− cells increased significantly in the 3D dynamic model compared with the 2D cultures. Expression of RUNX-2 was upregulated in the presence of purmorphamine in both cultures at day 14. The purmorphamine response gene, Gli-1, was upregulated during both the early and late culture periods in the 3D dynamic system, while similar up-regulation was observed only during the early culture period in 2D culture. No difference was observed in expressions of collagen type I and BSP between groups. The present study indicates that purmorphamine as agonist of Shh signaling pathway affects on osteogenic differentiation program of hMSCs in static and dynamic culture systems.