Adhesive Ligands Research Articles

Endothelial protein C receptor binding induces conformational changes to severe malaria-associated group A PfEMP1

Severe Plasmodium falciparum malaria infections are caused by microvascular sequestration of parasites binding to the human endothelial protein C receptor (EPCR) via the multi-domain P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands. Using cryogenic electron microscopy (Cryo-EM) and PfEMP1 sequence diversity analysis, we found that group A PfEMP1 CIDRα1 domains interact with the adjacent DBLα1 domain through central, conserved residues of the EPCR-binding site to adopt a compact conformation. Upon EPCR binding, the DBLα1 domain is displaced, and the EPCR-binding helix of CIDRα1 is turned, kinked, and twisted to reach a rearranged, stable EPCR-bound conformation. The unbound conformation and the required transition to the EPCR-bound conformation may represent a conformational masking mechanism of immune evasion for the PfEMP1 family.

Development of Mesoporous Silica Nanoparticle-Based Films with Tunable Arginine-Glycine-Aspartate Peptide Global Density and Clustering Levels to Study Stem Cell Adhesion and Differentiation.

Stem cell adhesion is mediated via the binding of integrin receptors to adhesion motifs present in the extracellular matrix (ECM). The spatial organization of adhesion ligands plays an important role in stem cell integrin-mediated adhesion. In this study, we developed a series of biointerfaces using arginine-glycine-aspartate (RGD)-functionalized mesoporous silica nanoparticles (MSN-RGD) to study the effect of RGD adhesion ligand global density (ligand coverage over the surface), spacing, and RGD clustering levels on stem cell adhesion and differentiation. To prepare the biointerface, MSNs were chemically functionalized with RGD peptides via an antifouling poly(ethylene glycol) (PEG) linker. The RGD surface functionalization ratio could be controlled to create MSNs with high and low RGD ligand clustering levels. MSN films with varying RGD global densities could be created by blending different ratios of MSN-RGD and non-RGD-functionalized MSNs together. A computational simulation study was performed to analyze nanoparticle distribution and RGD spacing on the resulting surfaces to determine experimental conditions. Enhanced cell adhesion and spreading were observed when RGD global density increased from 1.06 to 5.32 nmol cm-2 using highly clustered RGD-MSN-based films. Higher RGD ligand clustering levels led to larger cell spreading and increased formation of focal adhesions. Moreover, a higher RGD ligand clustering level promoted the expression of alkaline phosphatase in hMSCs. Overall, these findings indicate that both RGD global density and clustering levels are crucial variables in regulating stem cell behaviors. This study provides important information about ligand-integrin interactions, which could be implemented into biomaterial design to achieve optimal performance of adhesive functional peptides.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

Force-induced biphasic regulation of VWF cleavage by ADAMTS13

It is crucial for hemostasis that platelets are rapidly recruited to the site of vascular injury by the adhesive ligand von Willebrand factor (VWF) multimers. The metalloproteinase ADAMTS13 regulates this hemostatic activity by proteolytically reducing the size of VWF and its proteolytic kinetics has been investigated by biochemical and single-molecule biophysical methods. However, how ADAMTS13 cleaves VWF in flowing blood remains poorly defined. To investigate the force-induced VWF cleavage, VWF A1A2A3 tridomains were immobilized and subjected to hydrodynamic forces in the presence of ADAMTS13. We demonstrated that the cleavage of VWF A1A2A3 by ADAMTS13 exhibited biphasic kinetics governed by shear stress, but not shear rate. By fitting data to the single-molecule Michaelis-Menten equation, the proteolytic constant kcat of ADAMTS13 had two distinct states. The mean proteolytic constant of the fast state (kcat-fast) was 0.005 ± 0.001 s−1, which is >10-fold faster than the slow state (kcat-slow = 0.0005 ± 0.0001 s−1). Furthermore, proteolytic constants of both states were regulated by shear stress in a biphasic manner, independent of the solution viscosity, indicating that the proteolytic activity of ADAMTS13 was regulated by hydrodynamic force. The findings provide new insights into the mechanism underlying ADAMTS13 cleaving VWF under flowing blood.

Role of LIF genotypes on basal and day of triggering hormonal profile in women undergoing IUI

Female infertility is caused by ovulation issues, tubal disorders, uterine abnormalities, endometriosis, and peritoneal factors. For years, intrauterine insemination has been used to treat infertility alone or with ovarian stimulation. Obtaining adhesion ligands and losing inhibitory components are necessary for pregnancy. LIF promotes and differentiates proliferation and differentiation in embryonic, hematopoietic, and endothelial cells. LIF also controls prostaglandins (PGs), which mediate implantation and decidualization. The measuring hormone profile on cycle day 2 before control ovarian stimulation including FSH, LH, E2, Prolactin, TSH, and AMH. Serum LH, E2. The progesterone level was estimated on the day of triggering. The effect of LIF gene genotypes on hormone level. The hormone levels of 100 intrauterine inseminated women were measured on the second day of the cycle and the day of ovulation. DNA extraction for LIF gene variation detection utilizing sequencing and HRM. The current study found no significant differences between pregnant and non-pregnant groups for hormone levels, genotype, and genetic effect on female hormone levels with (P ≥0.05). The LIF gene did not affect hormone levels in infertile females may be due to the tiny samples or the complex signaling chain that happens during implantation

Engineering synthetic poly(ethylene) glycol-based hydrogels compatible with injection molding biofabrication.

Hydrogel injection molding is a biofabrication method that is useful for the rapid generation of complex cell-laden hydrogel geometries, with potential utility in biomanufacturing products for tissue engineering applications. Hydrogel injection molding requires that hydrogel polymers have sufficiently delayed crosslinking times to enable injection and molding prior to gelation. In this work, we explore the feasibility of injection molding synthetic poly(ethylene) glycol (PEG)-based hydrogels functionalized with strain promoted azide-alkyne cycloaddition click chemistry functional groups. We evaluate the mechanical properties of a PEG-based hydrogel library, including time to gelation and successful generation of complex geometries via injection molding. We evaluate the binding and retention of adhesive ligand RGD within the library matrices and characterize the viability and function of encapsulated cells. This work demonstrates the feasibility of injection molding synthetic PEG-based hydrogels for tissue engineering applications, with potential utility in the clinic and biomanufacturing.

Adhesion analysis via a tumor vasculature-like microfluidic device identifies CD8+ T cells with enhanced tumor homing to improve cell therapy

CD8+ Tcell recruitment to the tumor microenvironment is critical for the success of adoptive cell therapy (ACT). Unfortunately, only a small fraction of transferred cells home to solid tumors. Adhesive ligand-receptor interactions have been implicated in CD8+ Tcell homing; however, there is a lack of understanding of how CD8+ Tcells interact with tumor vasculature-expressed adhesive ligands under the influence of hemodynamic flow. Here, the capacity of CD8+ Tcells to home to melanomas is modeled exvivo using an engineered microfluidic device that recapitulates the hemodynamic microenvironment of the tumor vasculature. Adoptively transferred CD8+ Tcells with enhanced adhesion in flow invitroand tumor hominginvivo improve tumor control by ACT in combination with immune checkpoint blockade. These results show that engineered microfluidic devices can model the microenvironment of the tumor vasculature to identify subsets of Tcells with enhanced tumor infiltrating capabilities, a key limitation in ACT.

Electrostatic Assembly of Multiarm PEG-Based Hydrogels as Extracellular Matrix Mimics: Cell Response in the Presence and Absence of RGD Cell Adhesive Ligands.

Synthetic hydrogels have been used widely as extracellular matrix (ECM) mimics due to the ability to control and mimic physical and biochemical cues observed in natural ECM proteins such as collagen, laminin, and fibronectin. Most synthetic hydrogels are formed via covalent bonding resulting in slow gelation which is incompatible with drop-on-demand 3D bioprinting of cells and injectable hydrogels for therapeutic delivery. Herein, we developed an electrostatically crosslinked PEG-based hydrogel system for creating high-throughput 3D in vitro models using synthetic hydrogels to mimic the ECM cancer environment. A 3-arm PEG-based polymer backbone was first modified with either permanent cationic charged moieties (2-(methacryloyloxy)ethyl trimethylammonium) or permanent anionic charged moieties (3-sulfopropyl methacrylate potassium salt). The resulting charged polymers can be conjugated further with various amounts of cell adhesive RGD motifs (0, 25, 75, and 98%) to study the influences of RGD motifs on breast cancer (MCF-7) spheroid formation. Formation, stability, and mechanical properties of hydrogels were tested with, and without, RGD to evaluate the cellular response to material parameters in a 3D environment. The hydrogels can be degraded in the presence of salts at room temperature by breaking the interaction of oppositely charged polymer chains. MCF-7 cells could be released with high viability through brief exposure to NaCl solution. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Additionally, cell viability was not impacted by the tested hydrogel stiffness range between 60 to 1160 Pa. Taken together, this PEG-based tuneable hydrogel system shows great promise as a 3D ECM mimic of cancer extracellular environments with controllable biophysical and biochemical properties. The ease of gelation and dissolution through salt concentration provides a way to quickly harvest cells for further analysis at any given time of interest without compromising cell viability.

Towards the design of a conformational switchable zinc finger analogue regulating cellular behaviors.

Dynamic conformational changes in the secondary structures of proteins are essential to their functions and can regulate diverse cellular events. Herein, along with our experimental collaborators, we report the design of a synthetic polymer-based secondary structure analogue of Zinc finger (ZnF). Acting as a conformational switch between unfolded and folded states triggered by the addition of Zn2+ and EDTA, ZnF enables the manipulation of the accessibility of conjugated cell adhesive ligands to cell membrane receptors, by hiding or exposing the cell adhesive ligands to the transmembrane protein receptors. On the basis of the self-avoiding walk (SAW) model, we investigated the distribution of end-to-end distance of the polymer chain of ZnF and estimated the free energy barrier that the zinc coordination motif had to overcome, thereby, predicting the feasible length of the polymer chains. Our work provided valuable guidance to experimental research on the synthesis of biomimetic dynamic secondary structures to precisely control cell-biomaterial interactions and mediate the desired cellular behaviors.

Engineering viscoelastic alginate hydrogels for hiPSC cardiomyocyte culture.

Heart disease is the leading cause of death worldwide, however research progress of heritable cardiomyopathies has been limited by the lack of cost effective and accurate human models. Human induced pluripotent stem cells (hiPSCs) are a renewable cell source that can be differentiated into human cardiomyocytes (CMs), presenting a reliable tool for in vitro cellular models. The physiological environmental cues that CMs are exposed to are extraordinarily complex and in vitro systems typically fail to mimic key aspects such as viscoelasticity and adhesion ligand type. Viscoelasticity in particular is important to model as it is exhibited by virtually all tissues in the body and can influence several cell functions including spreading, differentiation and proliferation. However, the majority of experiments with CMs to date have used covalently crosslinked hydrogels that do not exhibit any stress relaxing properties. Here, we engineered biomaterials to recapitulate these viscoelastic heart tissue characteristics. Since the stress relaxation properties of heart tissue have not been directly measured, we first mechanically characterized left ventricular porcine tissue. The elastic modulus was around 7 kPa, within range of reported values. However, the tissue exhibited much faster stress relaxation values than we anticipated; relaxing to half its max value in less than 10 s while skeletal muscle tissue is reported to relax closer to 100 s. We synthesized an alginate hydrogel with conjugated PEG chains to mimic these fast-relaxing properties while maintaining similar elastic modulus values. We then seeded CMs on the hydrogels, imaged and analyzed the structure and function via confocal microscopy to assess cell adherence and morphology. These studies have validated our engineered hydrogel system as a hiPSC-CM culture platform and model system to investigate how viscoelasticity influences iPSC-CM function in wild-type and disease cell lines.

Ligand Coupling and Decoupling Modulates Stem Cell Fate

AbstractIn natural microenvironment, various proteins containing adhesive ligands in fibrous and non‐fibrous structures dynamically couple and decouple to regulate stem cell fate. Herein, materials presenting movably couplable ligands are developed by grafting liganded gold nanoparticles (AuNPs) to a substrate followed by flexibly grafting liganded movable linear nanomaterials (MLNs) to the substrate via a long bendable linker, thereby creating a space between the MLNs and the AuNPs in the decoupled state. Magnetic control of the MLNs decreases this space via the bending of the linker to couple the MLNs to the AuNPs. Remote control of ligand coupling stimulates integrin recruitment to the coupled ligands, thereby non‐toxically facilitating the focal adhesion, mechanosensing, and potential differentiation of stem cells, which is suppressed by ligand decoupling. Versatile tuning of size, aspect ratio, distributions, and ligands of the MLNs can help to decipher dynamic ligand‐coupling‐dependent stem cell fate to advance regenerative therapies.

Supercharged Protein Nanosheets for Cell Expansion on Bioemulsions.

Cell culture at liquid-liquid interfaces, for example, at the surface of oil microdroplets, is an attractive strategy to scale up adherent cell manufacturing while replacing the use of microplastics. Such a process requires the adhesion of cells at interfaces stabilized and reinforced by protein nanosheets displaying not only high elasticity but also presenting cell adhesive ligands able to bind integrin receptors. In this report, supercharged albumins are found to form strong elastic protein nanosheets when co-assembling with the co-surfactant pentafluorobenzoyl chloride (PFBC) and mediate extracellular matrix (ECM) protein adsorption and cell adhesion. The interfacial mechanical properties and elasticity of supercharged nanosheets are characterized by interfacial rheology, and behaviors are compared to those of native bovine serum albumin, human serum albumin, and α-lactalbumin. The impact of PFBC on such assembly is investigated. ECM protein adsorption to resulting supercharged nanosheets is then quantified via surface plasmon resonance and fluorescence microscopy, demonstrating that the dual role supercharged albumins are proposed to play as scaffold protein structuring liquid-liquid interfaces and substrates for the capture of ECM molecules. Finally, the adhesion and proliferation of primary human epidermal stem cells are investigated, at pinned droplets, as well as on bioemulsions stabilized by corresponding supercharged nanosheets. This study demonstrates the potential of supercharged proteins for the engineering of biointerfaces for stem cell manufacturing and draws structure-property relationships that will guide further engineering of associated systems.

Single-Cell Quantification of the Mechanical Stability of Cell-Cell Adherens Junction Using Glass Micropipettes.

Micropipette-based methods have been widely used for the manipulation of cells and characterization of the mechanical properties at the cell or tissue level. Here, we introduce the glass micropipette-based mechanical assays for the stability of cell-cell adhesion. A probing microbead coated with specific adhesion ligands, captured by a glass micropipette, is manipulated to form the adhesion complexes with the corresponding receptors on a single cell. Once the cell is moving away from the micropipette, forces are generated from 20 pN to 100 nN to the adhesion complexes, which are quantified in real-time based on the bending of the glass micropipette. We specifically emphasize the principle and method to probe the rupturing forces of the adhesion complexes at controlled force loading rates, the ligand coating on the probe microbeads, the force calibration of the glass micropipette, and the applications of the method to probe the E-cadherin-based cell-cell adhesions. The principles can be broadly applied to other cell adhesions such as cell-matrix adhesions, neuronal synapses, and bacterial-cell adhesions.

Stiffness-dependent active wetting enables optimal collective cell durotaxis

The directed migration of cellular clusters enables morphogenesis, wound healing and collective cancer invasion. Gradients of substrate stiffness direct the migration of cellular clusters in a process called collective durotaxis, but the underlying mechanisms remain unclear. Here we unveil a connection between collective durotaxis and the wetting properties of cellular clusters. We show that clusters of cancer cells dewet soft substrates and wet stiff ones. At intermediate stiffness—at the crossover from low to high wettability—clusters on uniform-stiffness substrates become maximally motile, and clusters on stiffness gradients exhibit optimal durotaxis. Durotactic velocity increases with cluster size, stiffness gradient and actomyosin activity. We demonstrate this behaviour on substrates coated with the cell–cell adhesion protein E-cadherin and then establish its generality on substrates coated with extracellular matrix. We develop an active wetting model that explains collective durotaxis in terms of a balance between in-plane active traction and tissue contractility and out-of-plane surface tension. Finally, we show that the distribution of cluster displacements has a heavy tail, with infrequent but large cellular hops that contribute to durotactic migration. Our study demonstrates a physical mechanism of collective durotaxis, through both cell–cell and cell–substrate adhesion ligands, based on the wetting properties of active droplets. Substrate stiffness influences cellular cluster migration through collective durotaxis. Now, the underlying mechanism of this process is explained by considering the wetting dynamics of the clusters.

Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity.

Various signals in tissue microenvironments are often unevenly distributed around cells. Cellular responses to asymmetric cell-matrix adhesion in a 3D space remain generally unclear and are to be studied at the single-cell resolution. Here, the authors developed a droplet-based microfluidic approach to manufacture a pure population of single cells in a microscale layer of compartmentalized 3D hydrogel matrices with a tunable spatial presentation of ligands at the subcellular level. Cells elongate with an asymmetric presentation of the integrin adhesion ligand Arg-Gly-Asp (RGD), while cells expand isotropically with a symmetric presentation of RGD. Membrane tension is higher on the side of single cells interacting with RGD than on the side without RGD. Finite element analysis shows that a non-uniform isotropic cell volume expansion model is sufficient to recapitulate the experimental results. At a longer timescale, asymmetric ligand presentation commits mesenchymal stem cells to the osteogenic lineage. Cdc42 is an essential mediator of cell polarization and lineage specification in response to asymmetric cell-matrix adhesion. This study highlights the utility of precisely controlling 3D ligand presentation around single cells to direct cell polarity for regenerative engineering and medicine.

Directed Conformational Switching of a Zinc Finger Analogue Regulates the Mechanosensing and Differentiation of Stem Cells.

The dynamic conformational changes in the secondary structures of proteins are essential to their functions and can regulate diverse cellular events. Herein we report the design of a synthetic polymer-based secondary structure analogue of a zinc finger (ZnF) by introducing a zinc coordination motif to overcome the free energy barrier predicted by theoretical calculations and fold-free polymer chains. The conformational switching between unfolded and folded state of the ZnF analogue can be triggered in situ to drastically manipulate the accessibility of conjugated cell adhesive ligands to the cell membrane receptors, thereby effectively controlling the adhesion, spreading, mechanosensing, and differentiation of stem cells. We believe that emulating the dynamic secondary structures of proteins via rational design of a folded synthetic polymer-cation complex is a promising strategy for developing bioactive materials to mediate desired cellular functions.

Directed Conformational Switching of a Zinc Finger Analogue Regulates the Mechanosensing and Differentiation of Stem Cells

The dynamic conformational changes in the secondary structures of proteins are essential to their functions and can regulate diverse cellular events. Herein we report the design of a synthetic polymer-based secondary structure analogue of a zinc finger (ZnF) by introducing a zinc coordination motif to overcome the free energy barrier predicted by theoretical calculations and fold-free polymer chains. The conformational switching between unfolded and folded state of the ZnF analogue can be triggered in situ to drastically manipulate the accessibility of conjugated cell adhesive ligands to the cell membrane receptors, thereby effectively controlling the adhesion, spreading, mechanosensing, and differentiation of stem cells. We believe that emulating the dynamic secondary structures of proteins via rational design of a folded synthetic polymer–cation complex is a promising strategy for developing bioactive materials to mediate desired cellular functions.

Role of stiffness and physico-chemical properties of tumour microenvironment on breast cancer cell stemness

Several physico-chemical properties of the tumour microenvironment (TME) are dysregulated during tumour progression, such as tissue stiffness, extracellular pH and interstitial fluid flow. Traditional preclinical models, although useful to study biological processes, do not provide sufficient control over these physico-chemical properties, hence limiting the understanding of cause-effect relationships between the TME and cancer cells. Breast cancer stem cells (B-CSCs), a dynamic population within the tumour, are known to affect tumour progression, metastasis and therapeutic resistance. With their emerging importance in disease physiology, it is essential to study the interplay between above-mentioned TME physico-chemical variables and B-CSC marker expression. In this work, 3D in vitro models with controlled physico-chemical properties (hydrogel stiffness and composition, perfusion, pH) were used to mimic normal and tumour breast tissue to study changes in proliferation, morphology and B-CSC population in two separate breast cancer cell lines (MCF-7 and MDA-MB 231). Cells encapsulated in alginate-gelatin hydrogels varying in stiffness (2-10 kPa), density and adhesion ligand (gelatin) were perfused (500 µL/min) for up to 14 days. Physiological (pH 7.4) and tumorigenic (pH 6.5) media were used to mimic changes in extracellular pH within the TME. We found that both cell lines have distinct responses to changes in physico-chemical factors in terms of proliferation, cell aggregates size and morphology. Most importantly, stiff and dense hydrogels (10 kPa) and acidic pH (6.5) play a key role in B-CSCs dynamics, increasing both epithelial (E-CSCs) and mesenchymal cancer stem cell (M-CSCs) marker expression, supporting direct impact of the physico-chemical microenvironment on disease onset and progression. Statement of significanceCurrently no studies evaluate the impact of physico-chemical properties of the tumour microenvironment on breast cancer stem cell (B-CSC) marker expression in a single in vitro model and at the same time. In this study, 3D in vitro models with varying stiffness, extracellular pH and fluid flow are used to recapitulate the breast tumour microenvironment to evaluate for the first time their direct effect on multiple breast cancer phenotypes: cell proliferation, cell aggregate size and shape, and B-CSC markers. Results suggest these models could open new ways of monitoring disease phenotypes, from the early-onset to progression, as well as being used as testing platforms for effective identification of specific phenotypes in the presence of relevant tumour physico-chemical microenvironment.

Customized Multifunctional Peptide Hydrogel Scaffolds for CAR-T-Cell Rapid Proliferation and Solid Tumor Immunotherapy.

CAR-T-cell therapies must be expanded to obtain a large number of effector cells quickly, and the current technology cannot address this challenge. A longer operational time would lose or alter the function and phenotype of CAR-T cells in response to therapy, and it also causes a loss in the optimal treatment time for patients. At present, lower survival time and homing efficiency reduce the antitumor effect of CAR-T in vivo. But nobody has solved these two issues in one system, which has a similar microenvironment of lymphoid organs to activate/expand cell delivery for immunotherapy. Here, we generated artificial, customized immune cell matrix scaffolds based on a self-assembling peptide to preserve and augment the cell phenotype in light of the characteristics of CAR-T. The all-in-one nanoscale matrix scaffolds reduced the processing time of CAR-T to 3 days and resulted in over a 10-fold increase compared with the traditional protocol. The cells were combined to modulate mechanotransduction and chemical signals, and the mimic matrix scaffolds showed optimal stiffness and adhesive ligand density, thereby accelerating CAR-T-cell proliferation. Meanwhile, engineering CAR-T-secreted intrinsic PD-1 blocking single-chain variable fragments (scFv) further increased cell proliferation and cytotoxicity by resisting the self and tumor microenvironment in a paracrine and autocrine manner. Local delivery of CAR-T cells from the scaffolds significantly enabled long-term retention, suppressed tumor growth, and increased infiltration of effector T cells compared with traditional CAR-T treatment. The application of bioengineering and genetic engineering approaches has led to the development of rapid culture environments that can control matrix scaffold properties for CAR-T-cell and cancer immunotherapies.

Construction of polysaccharide scaffold-based perfusion bioreactor supporting liver cell aggregates for drug screening

Rebuilding a suitable microenvironment of liver cells is the key challenge to enhancing the expression of hepatic functions for drug screening in vitro. To improve the microenvironment by providing the specific adhesive ligands for hepatocytes in the three-dimensional dynamic culture, a perfusion bioreactor with a pectin/alginate blend porous scaffold was constructed in this study. The galactosyl component in the main chain of pectin was able to be specifically recognized by the asialoglycoprotein receptor on the surface of hepatocytes, and subsequently promoted the adhesion and aggregation of hepatocytes co-cultured with hepatic non-parenchymal cells. The bioreactor was optimized for 4 h of dynamic inoculation followed by perfusion at a flow rate of 2 mL/min, which provided adequate oxygen supply and good mass transfer to the liver cells. During dynamic cultured in the bioreactor for 14 days, more multicellular aggregates were formed and were evenly distributed in the pectin/alginate blend scaffolds. The expressions of intercellular interaction and hepatic functions of the hepatocytes in aggregates were significantly enhanced in the three-dimensional dynamic group. Furthermore, the bioreactor not only markedly upregulated the cell polarity markers expression of hepatocytes but also enhanced their metabolic capacity to acetaminophen, isoniazid, and tolbutamide, which exhibited a significant concentration-dependent manner. Therefore, the pectin/alginate blend scaffold-based perfusion bioreactor appeared to be a promising candidate in the field of drug development and liver regeneration research.