Integration of 3D bioprinting and multi-algorithm machine learning identified glioma susceptibilities and microenvironment characteristics.

Glioma, with its heterogeneous microenvironments and genetic subtypes, presents substantial challenges for treatment prediction and development. We integrated 3D bioprinting and multi-algorithm machine learning as a novel approach to enhance the assessment and understanding of glioma treatment responses and microenvironment characteristics. The bioprinted patient-derived glioma tissues successfully recapitulated molecular properties and drug responses of native tumors. We then developed GlioML, a machine learning workflow incorporating nine distinct algorithms and a weighted ensemble model that generated robust gene expression-based predictors, each reflecting the diverse action mechanisms of various compounds and drugs. The ensemble model superseded the performance of all individual algorithms across diverse in vitro systems, including sphere cultures, complex 3D bioprinted multicellular models, and 3D patient-derived tissues. By integrating bioprinting, the evaluative scope of the treatment expanded to T cell-related therapy and anti-angiogenesis targeted therapy. We identified promising compounds and drugs for glioma treatment and revealed distinct immunosuppressive or angiogenic myeloid-infiltrated tumor microenvironments. These insights pave the way for enhanced therapeutic development for glioma and potentially for other cancers, highlighting the broad application potential of this integrative and translational approach.

Advances in the bladder cancer research using 3D culture models.

Bladder cancer represents the most common malignancy of the urinary system, posing a significant threat to patients' life. Animal models and two-dimensional (2D) cell cultures, among other traditional models, have been used for years to study various aspects of bladder cancer. However, these methods are subject to various limitations when mimicking the tumor microenvironment in vivo, thus hindering the further improvement of bladder cancer treatments. Recently, three-dimensional (3D) culture models have attracted extensive attention since they overcome the shortcomings of their traditional counterparts. Most importantly, 3D culture models more accurately reproduce the tumor microenvironment in the human body because they can recapitulate the cell-cell and cell-extracellular matrix interactions. 3D culture models can thereby help us gain deeper insight into the bladder cancer. The 3D culture models of tumor cells can extend the culture duration and allow for co-culturing with different cell types. Study of patient-specific bladder cancer mutations and subtypes is made possible by the ability to preserve cells isolated from particular patients in 3D culture models. It will be feasible to develop customized treatments that target relevant signaling pathways or biomarkers. This article reviews the development, application, advantages, and limitations of traditional modeling systems and 3D culture models used in the study of bladder cancer and discusses the potential application of 3D culture models.

Machine Learning Assisted Wearable Wireless Device for Sleep Apnea Syndrome Diagnosis.

Sleep apnea syndrome (SAS) is a common but underdiagnosed health problem related to impaired quality of life and increased cardiovascular risk. In order to solve the problem of complicated and expensive operation procedures for clinical diagnosis of sleep apnea, here we propose a small and low-cost wearable apnea diagnostic system. The system uses a photoplethysmography (PPG) optical sensor to collect human pulse wave signals and blood oxygen saturation synchronously. Then multiscale entropy and random forest algorithms are used to process the PPG signal for analysis and diagnosis of sleep apnea. The SAS determination is based on the comprehensive diagnosis of the PPG signal and blood oxygen saturation signal, and the blood oxygen is used to exclude the error induced by non-pathological factors. The performance of the system is compared with the Compumedics Grael PSG (Polysomnography) sleep monitoring system. This simple diagnostic system provides a feasible technical solution for portable and low-cost screening and diagnosis of SAS patients with a high accuracy of over 85%.

Design and aligner-assisted fast fabrication of a microfluidic platform for quasi-3D cell studies on an elastic polymer.

While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D. Herein, we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip. The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers. To resolve the problem, we designed and assembled a fast aligner that is cost-effective and convenient to operate. To enable quasi-3D microenvironment of cells, we fabricated polydimethylsiloxane (PDMS) microwell arrays (formed by micropillars of a few microns in diameter) with the microwell diameters close to the cell sizes. An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS. We examined three types of cells in 2D, quasi-3D, and 3D microenvironments; the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells. We also constructed transgenic human mesenchymal stem cells (hMSCs); under cyclic stretching, the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate. This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies, but also inspires further studies of the topological effects of biomaterials on cells.

Critical Frequency and Critical Stretching Rate for Reorientation of Cells on a Cyclically Stretched Polymer in a Microfluidic Chip.

The ability of cells to sense and respond to mechanical signals from their surrounding microenvironments is one of the key issues in tissue engineering and regeneration, yet a fundamental study of cells with both cell observation and mechanical stimulus is challenging and should be based upon an appropriate microdevice. Herein we designed and fabricated a two-layer microfluidic chip to enable simultaneous observation of live cells and cyclic stretching of an elastic polymer, polydimethylsiloxane (PDMS), with a modified surface for enhanced cell adhesion. Human mesenchymal stem cells (hMSCs) were examined with a series of frequencies from 0.00003 to 2 Hz and varied amplitudes of 2%, 5%, or 10%. The cells with an initial random orientation were confirmed to be reoriented perpendicular to the stretching direction at frequencies greater than a threshold value, which we term critical frequency (fc); additionally, the critical frequency fc was amplitude-dependent. We further introduced the concept of critical stretching rate (Rc) and found that this quantity can unify both frequency and amplitude dependences. The reciprocal value of Rc in this study reads 8.3 min, which is consistent with the turnover time of actin filaments reported in the literature, suggesting that the supramolecular relaxation in the cytoskeleton within a cell might be responsible for the underlying cell mechanotransduction. The theoretical calculation of cell reorientation based on a two-dimensional tensegrity model under uniaxial cyclic stretching is well consistent with our experiments. The above findings provide new insight into the crucial role of critical frequency and critical stretching rate in regulating cells on biomaterials under biomechanical stimuli.

A simplified yet enhanced and versatile microfluidic platform for cyclic cell stretching on an elastic polymer.

While the microfluidic chips for cell stretching and real-time cell observations have so far been composed of three layers, the present work reports a two-layer one, which is, on the surface, not available due to the 'inherent' difficulty of unstable focusing on cells in the microscopic observation under the stretching operation, etc. Herein, this difficulty was overcome to a large extent, in the case of appropriate device parameters, which were determined based upon finite element analysis and orthogonal experimental design. The novel chip was fabricated and confirmed to work in frequency up to 2 Hz and stretching ratio up to 20%. We further performed uniaxial stretching experiments of human mesenchymal stem cells on an elastic polymer, polydimethylsiloxane, and the cells were found to be highly oriented perpendicular to the stretching direction. The short working distance on this simplified two-layer chip enabled clear observation of microtubules and stress fibers of cells under an optical microscope. We also tested radial stretching and gradient stretching as proofs of concept of the extendibility of this type of chip. Therefore, in spite of being simpler, the two-layer chip suggested in this study exhibited enhanced and versatile functions, and the present work has thus afforded a new methodology of fabrication of microfluidic chips for the study of cells on biomaterials under a mechanical stimulus.

Cell migration regulated by RGD nanospacing and enhanced under moderate cell adhesion on biomaterials.

While nanoscale modification of a biomaterial surface is known to influence various cell behaviors, it is unclear whether there is an optimal nanospacing of a bioactive ligand with respect to cell migration. Herein, we investigated the effects of nanospacing of arginine-glycine-aspartate (RGD) peptide on cell migration and its relation to cell adhesion. To this end, we prepared RGD nanopatterns with varied nanospacings (31-125 nm) against the nonfouling background of poly(ethylene glycol), and employed human umbilical vein endothelial cells (HUVECs) to examine cell behaviors on the nanopatterned surfaces. While HUVECs adhered well on surfaces of RGD nanospacing less than 70 nm and exhibited a monotonic decrease of adhesion with the increase of RGD nanospacing, cell migration exhibited a nonmonotonic change with the ligand nanospacing: the maximum migration velocity was observed around 90 nm of nanospacing, and slow or very slow migration occurred in the cases of small or large RGD nanospacings. Therefore, moderate cell adhesion is beneficial for fast cell migration. Further molecular biology studies revealed that attenuated cell adhesion and activated dynamic actin rearrangement accounted for the promotion of cell migration, and the genes of small G proteins such as Cdc42 were upregulated correspondingly. The present study sheds new light on cell migration and its relation to cell adhesion, and paves a way for designing biomaterials for applications in regenerative medicine.

Nonmonotonic Self-Deformation of Cell Nuclei on Topological Surfaces with Micropillar Array.

Cells respond to the mechanical signals from their surroundings and integrate physiochemical signals to initiate intricate mechanochemical processes. While many studies indicate that topological features of biomaterials impact cellular behaviors profoundly, little research has focused on the nuclear response to a mechanical force generated by a topological surface. Here, we fabricated a polymeric micropillar array with an appropriate dimension to induce a severe self-deformation of cell nuclei and investigated how the nuclear shape changed over time. Intriguingly, the nuclei of mesenchymal stem cells (MSCs) on the poly(lactide-co-glycolide) (PLGA) micropillars exhibited a significant initial deformation followed by a partial recovery, which led to an "overshoot" phenomenon. The treatment of cytochalasin D suppressed the recovery of nuclei, which indicated the involvement of actin cytoskeleton in regulating the recovery at the second stage of nuclear deformation. Additionally, we found that MSCs exhibited different overshoot extents from their differentiated lineage, osteoblasts. These findings enrich the understanding of the role of the cell nucleus in mechanotransduction. As the first quantitative report on nonmonotonic kinetic process of self-deformation of a cell organelle on biomaterials with unique topological surfaces, this study sheds new insight into cell-biomaterial interactions.

Subcellular cell geometry on micropillars regulates stem cell differentiation.

While various material factors have been shown to influence cell behaviors, recent studies started to pay attention to the effects of some material cues on "subcellular" geometry of cells, such as self-deformation of cell nuclei. It is particularly interesting to examine whether a self deformation happens discontinuously like a first-order transition and whether subcellular geometry influences significantly the extent of stem cell differentiation. Herein we prepared a series of micropillar arrays of poly(lactide-co-glycolide) and discovered a first-order transition of nuclear shape as a function of micropillar height under the examined section area and interspacing of the pillars. The deformed state of the nuclei of mesenchymal stem cells (MSCs) was well maintained even after osteogenic or adipogenic induction for several days. The nuclear deformation on the micropillar arrays was accompanied with smaller projected areas of cells, but led to an enhanced osteogenesis and attenuated adipogenesis of the MSCs, which is different from the previously known relationship between morphology and differentiation of stem cells on flat substrates. Hence, the present study reveals that the geometry of cell nuclei may afford a new cue to regulate the lineage commitment of stem cells on the subcellular level.

Ex vivo 3D osteocyte network construction with primary murine bone cells.

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Microbeads-Guided Reconstruction of 3D Osteocyte Network during Microfluidic Perfusion Culture.

Osteocytes reside as 3-dimensionally networked cells in the lacunocanalicular structure of bones, and function as the master regulators of homeostatic bone remodeling. We report here, for the first time to our best knowledge, the use of a biomimetic approach to reconstruct the 3D osteocyte network with physiological relevant microscale dimensions. In this approach, biphasic calcium phosphate microbeads were assembled with murine early osteocytes (MLO-A5) to provide an initial mechanical framework for 3D network formation and maintenance during long-term perfusion culture in a microfluidic chamber. The microbead size of 20-25 µm was used to: (1) facilitate a single cell to be placed within the interstitial space between the microbeads, (2) mitigate the proliferation of the entrapped cell due to its physical confinement in the interstitial site, and (3) control cell-to-cell distance to be 20-25 µm as observed in murine bones. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads within 3 days of culture. The entrapped cells produced significant mineralized extracellular matrix to fill up the interstitial spaces, resulting in the formation of a dense tissue structure during the course of 3-week culture. We found that the time-dependent osteocytic transitions of the cells exhibited trends consistent with in vivo observations, particularly with high expression of Sost gene, which is a key osteocyte-specific marker for the mechanotransduction function of osteocytes. In contrast, cells cultured in 2D well-plates did not replicate in vivo trends. These results provide an important new insight in building physiologically relevant in vitro bone tissue models.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Chitosan surface enhances the mobility, cytoplasm spreading, and phagocytosis of macrophages.

A chitosan micropattern was prepared on glass by inkjet printing to visualize and compare in real-time macrophage developments on chitosan versus glass during microfluidic culture. The mobility of macrophages on chitosan was significantly higher, since the cells on glass were anchored by the development of podosomes whereas those on chitosan did not form podosomes. The phagocytosis of bacteria by macrophages was considerably more effective on chitosan because of: (1) the macrophages' higher mobility to scavenge nearby bacteria and (2) their cyotoplasm's ability to spread, re-distribute, and recover more freely to engulf the bacteria. Consequently, bacteria growth on chitosan surface was significantly reduced in the presence of macrophages in comparison to that on glass surface, as measured by surface bacteria density and effluent bacteria concentration. These findings suggest the synergistic effect of chitosan as a potential coating material on biomedical implants in promoting macrophage response upon the arrival of opportunistic bacteria.

Inkjet printed antibiotic- and calcium-eluting bioresorbable nanocomposite micropatterns for orthopedic implants.

Inkjet printing of antibiotic- and calcium-eluting micropatterns was explored as a novel means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on orthopedic implant surfaces. The micropatterns consisted of a periodic array of ∼50 µm circular dots separated by ∼150 µm. The composition of the micropatterns was controlled by formulating inks with rifampicin (RFP) and poly(D,L-lactic-co-glycolic) acid (PLGA) dissolved in an organic solvent with ∼100 nm biphasic calcium phosphate (BCP) nanoparticles suspended in the solution. During printing RFP and PLGA co-precipitated to form a nanocomposite structure with ∼10-100 nm RFP and the BCP particles dispersed in the PLGA matrix. The rate of RFP release was strongly influenced by the RFP loading in the micropattern, particularly on the first day. The RFP-containing micropatterns effectively prevented the formation of Staphylococcus epidermidis biofilm colonies due to their ability to kill bacteria prior to forming colonies on the patterned surfaces. The BCP-containing micropatterns printed on the surface of the alloy TiAl6V4 significantly accelerated osteoblast cell differentiation, as measured by alkaline phosphatase expression and calcium deposition, without compromising cell proliferation.

Microfluidic 3D bone tissue model for high-throughput evaluation of wound-healing and infection-preventing biomaterials.

We report the use of a microfluidic 3D bone tissue model, as a high-throughput means of evaluating the efficacy of biomaterials aimed at accelerating orthopaedic implant-related wound-healing while preventing bacterial infection. As an example of such biomaterials, inkjet-printed micropatterns were prepared to contain antibiotic and biphasic calcium phosphate (BCP) nanoparticles dispersed in a poly(D,L-lactic-co-glycolic) acid matrix. The micropatterns were integrated with a microfluidic device consisting of eight culture chambers. The micropatterns immediately and completely killed Staphylococcus epidermidis upon inoculation, and enhanced the calcified extracellular matrix production of osteoblasts. Without antibiotic elution, bacteria rapidly proliferated to result in an acidic microenvironment which was detrimental to osteoblasts. These results were used to demonstrate the tissue model's potential in: (i) significantly reducing the number of biomaterial samples and culture experiments required to assess in vitro efficacy for wound-healing and infection prevention and (ii) in situ monitoring of dynamic interactions of biomaterials with bacteria as wells as with tissue cells simultaneously.