Cellular and molecular bioengineering最新文献

Purpose

Affibodies are a class of versatile affinity proteins with a wide variety of therapeutic applications, ranging from contrast agents for imaging to cell-targeting therapeutics. We have identified several affibodies specific to bone morphogenetic protein-2 (BMP-2) with a range of binding affinities and demonstrated the ability to tune release rate of BMP-2 from affibody-conjugated poly(ethylene glycol) maleimide (PEG-mal) hydrogels based on affibody affinity strength. In this work, we compare the purity, structure, and activity of recombinant, bacterially-expressed BMP-2-specific affibodies with affibodies synthesized via solid-phase peptide synthesis.

Methods

High- and low-affinity BMP-2-specific affibodies were recombinantly expressed using BL21(DE3) E. coli and chemically synthesized using microwave-assisted solid-phase peptide synthesis with Fmoc-Gly-Wang resin. The secondary structures of the affibodies and dissociation constants of affibody-BMP-2 binding were characterized by circular dichroism and biolayer interferometry, respectively. Endotoxin levels were measured using chromogenic limulus amebocyte lysate (LAL) assays. Affibody-conjugated PEG-mal hydrogels were fabricated and loaded with BMP-2 to evaluate hydrogel capacity for controlled release, quantified by enzyme-linked immunosorbent assays (ELISA).

Results

Synthetic and recombinant affibodies were determined to be α-helical by circular dichroism. The synthetic high- and low-affinity BMP-2-specific affibodies demonstrated comparable BMP-2 binding dissociation constants to their recombinant counterparts. Recombinant affibodies retained some endotoxins after purification, while endotoxins were not detected in the synthetic affibodies above FDA permissible limits. High-affinity affibody-conjugated hydrogels reduced cumulative BMP-2 release compared to the low-affinity affibody-conjugated hydrogels and hydrogels without affibodies.

Conclusions

Synthetic affibodies demonstrate comparable structure and function to recombinant affibodies while reducing endotoxin contamination and increasing product yield, indicating that solid-phase peptide synthesis is a viable method of producing affibodies for controlled protein release and other applications.

Purpose

Accelerating wound healing is a main consideration in surgery. The three stages of wound healing are inflammatory response, tissue repair and cell proliferation. Much research has focused on epidermal cell proliferation and migration because this is an essential step in wound healing.

Methods and Results

The current study discovered that exosomes from Adipose-derived stem cell (ADSC) following hypoxic preconditioning (HExo) have a greater promotional effect on vaginal wound healing. Protein kinase B (AKT)/hypoxia-inducible factor 1-alpha (HIF-1α) play an important role in HExo-mediated HaCaT cell migration and proliferation. The promotional effect of HExo on rat wound healing was reversed by both, HIF‑1α and AKT inhibition. Phosphorylation of AKT (p-AKT) or HIF‑1α suppression reversed the protective effect of HExo on vaginal wound healing.

Conclusion

Taken together, our study found that hypoxic preconditioning of adipose MSC exosomes enhances vaginal wound healing via accelerated keratinocyte proliferation and migration through AKT/HIF‑1α axis activation.

Purpose

The bidirectional regulation of macrophages and exosomes provides a meaningful research direction for the treatment of complications arising from both type 1 and type 2 diabetes mellitus. However, there is currently no comprehensive evaluation of the bidirectional regulatory role of macrophages and exosomes in diabetic complications. In this review, we aim to provide the detailed process of the bidirectional regulation mechanism of macrophages and exosomes, and how macrophage-associated exosomes use this mechanism to make it better applied to clinical practice through biotechnology.

Methods

Therefore, we summarized the bidirectional regulation mechanism of macrophages and exosomes and the application based on the bidirectional regulation mechanism from two aspects of inflammation and insulin resistance.

Results

As key regulators of the immune system, macrophages are crucial in the progression of diabetic complications due to their significant impact on the regulation of cellular metabolism, inflammation, and insulin sensitivity. Furthermore, exosomes, as innovative mediators of intercellular communication, transport miRNAs, proteins, and various bioactive molecules, influencing the occurrence and progression of diabetic complications through the regulation of inflammation and insulin resistance. The bidirectional regulation between macrophages and exosomes provides a promising pathway for the treatment of diabetic complications aimed at regulating the immune response and improving insulin sensitivity.

Conclusions

Understanding the complexity of the interaction between macrophages and exosomes can advance the treatment of diabetic complications and drug development, and bringing more innovative and effective treatment strategies for diabetic complications.

Purpose

Macrophage immune cells play crucial roles in the inflammatory (M1) and regenerative (M2) processes. The extracellular matrix (ECM) composition, including presentation of embedded ligands, governs macrophage function. Laminin concentration is abundant in the basement membrane and is dependent on pathological state: reduced in inflammation and increased during regeneration. Distinct laminin ligands, such as IKVAV and YIGSR, have disparate roles in dictating cell function. For example, IKVAV, derived from the alpha chain of laminin, promotes angiogenesis and metastasis of cancer cells whereas YIGSR, beta chain derived, impedes angiogenesis and tumor progression. Previous work has demonstrated IKVAV’s inflammation inhibiting properties in macrophages. Given the divergent role of IKVAV and YIGSR in interacting with cells through varied integrin receptors, we ask: what role does laminin derived peptide YIGSR play in governing macrophage function?

Methods

We quantified the influence of YIGSR on macrophage phenotype in 2D and 3D via immunostaining assessments for M1 marker inducible nitric oxide synthase (iNOS) and M2 marker Arginase−1 (Arg-1). We also analysed the secretome of human and murine macrophage response to YIGSR via a Luminex bead assay.

Results

YIGSR impact on macrophage phenotype occurs in a concentration-dependent manner. At lower concentrations of YIGSR, macrophage inflammation was increased whereas, at higher concentrations of YIGSR the opposite effect was seen within the same time frame. Secretomic assessments also demonstrate that pro-inflammatory chemokines and cytokines were increased at low YIGSR concentrations in M0, M1, M2 macrophages while pro-inflammatory secretion was reduced at higher concentrations.

Conclusions

YIGSR can be used as a tool to modulate macrophage inflammatory state within M1 and M2 phenotypes depending on the concentration of peptide. YIGSR’s impact on macrophage function can be leveraged for the development of immunoengineering strategies in regenerative medicine and cancer therapy.

Purpose

The suboptimal clinical performance of human mesenchymal stem cells (hMSCs) has raised concerns about their therapeutic potential. One major contributing factor to this issue is the heterogeneous nature of hMSCs. Senescent cell accumulation during stem cell expansion is a key driver of MSC heterogeneity. Current methodologies to eradicate senescent hMSCs have either shown limited success or lack clinical relevance. This study leverages the inherent capacity of hMSCs to migrate toward damaged tissues as a means to discern senescent from presenescent stem cells. Given the established deficiency of senescent cells to migrate through physiologically relevant environments, we hypothesized that a microfluidic device, designed to emulate key facets of in vivo cell motility, could serve as a platform for identifying presenescent cells.

Methods

We employed a Y-shaped microchannel assay, which allows fine-tuning of fluid flow rates and the degree of confinement.

Results

Highly migratory hMSCs detected by the device not only demonstrate increased speed, smaller size, and higher proliferative capacity but also manifest reduced DNA damage and senescence compared to non-migratory cells. Additionally, this assay detects presenescent cells in experiments with mixed early and late passage cells. The introduction of fluid flow through the device can further increase the fraction of highly motile stem cells, improving the assay's effectiveness to remove senescent hMSCs.

Conclusions

Collectively, this assay facilitates the detection and isolation of a highly potent stem cell subpopulation. Given the positive correlation between the migratory potential of administered MSCs and the long-term clinical outcome, delivering homogeneous, highly motile presenescent hMSCs may benefit patient outcomes.

Introduction

Dysregulated neutrophil function plays a significant role in the pathology of infections, cancer, cardiovascular diseases, and autoimmune disorders. Neutrophil activity is influenced by various cell populations, including macrophages, which are crucial regulators. However, the exact role of human macrophages in controlling neutrophil function remains unclear due to a scarcity of studies utilizing human cells in physiologically relevant models.

Methods

We adapted our “Infection-on-a-Chip” microfluidic device to incorporate macrophages within the collagen extracellular matrix, allowing for the study of interactions between human neutrophils and macrophages in a context that mimics in vivo conditions. The integration of THP-1 macrophages was optimized and their effect on the endothelial lumen was characterized, focusing on permeability and structural integrity. The device was then employed to examine the influence of macrophages on neutrophil response to infection with the bacterial pathogen Pseudomonas aeruginosa.

Results

Integration of THP-1 macrophages into the microfluidic device was successfully optimized, showing no increase in endothelial permeability or structural damage. The presence of macrophages was found to significantly reduce neutrophil transendothelial migration in response to Pseudomonas aeruginosa infection.

Conclusions

Our findings highlight the regulatory role of macrophages in modulating neutrophil responses, suggesting potential therapeutic targets to control neutrophil function in various diseases. The modified microfluidic platform offers a valuable tool for mechanistic studies into macrophage-neutrophil interactions in disease contexts.

Introduction

Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress.

Methods

We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity.

Results

NK cells had heterogeneous killing activity during 10 h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing.

Conclusions

Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data.

Introduction

Multidomain peptides (MDPs) are amino acid sequences that self-assemble to form supramolecular hydrogels under physiological conditions that have shown promise for a number of biomedical applications. K₂(SL)₆K₂ (“K₂”), a widely studied MDP, has demonstrated the ability to enhance the humoral immune response to co-delivered antigen. Herein, we sought to explore the in vitro and in vivo properties of a peptide with the same sequence but opposite chirality (D-K₂) since peptides composed of D-amino acids are resistant to protease degradation and potentially more immunostimulatory than their canonical counterparts.

Methods

K₂ and D-K₂ hydrogels were characterized and evaluated in vitro using circular dichroism, rheology, cryo-electron microscopy, and fluorescence recovery after photobleaching studies. In vivo experiments in SKH-1 mice were conducted to evaluate both ovalbumin release from the hydrogels and hydrogel degradation. The injection site of the hydrogels was analyzed using histology and humoral immunity was assessed by ELISA.

Results

In vitro, the enantiomeric hydrogels exhibited similar rheological properties, and fluorescence recovery after photobleaching experiments demonstrated that the diffusion of ovalbumin (OVA), a model antigen, was similar within both hydrogels. In vivo, K₂ and D-K₂ peptide hydrogels had similar OVA release rates, both releasing 89% of the antigen within 8 days. Both hydrogels elicited a similar antigen-specific humoral immune response. However, the in vivo degradation of the D-K₂ hydrogel progressed significantly slower than K₂. After 4 weeks in vivo, only 23 ± 7% of the K₂ hydrogel remained at the injection site compared to 94 ± 7% of the D-K₂ hydrogel, likely due to their different protease susceptibilities.

Conclusion

Taken together, these data suggest that peptide chirality can be a useful tool for increasing hydrogel residence time for biomedical applications that would benefit from long persistence times and that, if an antigen releases over a sufficiently short period, release can be largely independent of degradation rate, though slower-diffusing payloads may exhibit degradation rate dependence.

Introduction

Mechanical forces provide critical biological signals to cells. Within the distal lung, tensile forces act across the basement membrane and epithelial cells atop. Stretching devices have supported studies of mechanical forces in distal lung epithelium to gain mechanistic insights into pulmonary diseases. However, the integration of curvature into devices applying mechanical forces onto lung epithelial cell monolayers has remained challenging. To address this, we developed a hammock-shaped platform that offers desired curvature and mechanical forces to lung epithelial monolayers.

Methods

We developed hammocks using polyethylene terephthalate (PET)-based membranes and magnetic-particle modified silicone elastomer films within a 48-well plate that mimic the alveolar curvature and tensile forces during breathing. These hammocks were engineered and characterized for mechanical and cell-adhesive properties to facilitate cell culture. Using human small airway epithelial cells (SAECs), we measured monolayer formation and mechanosensing using F-Actin staining and immunofluorescence for cytokeratin to visualize intermediate filaments.

Results

We demonstrate a multi-functional design that facilitates a range of curvatures along with the incorporation of magnetic elements for dynamic actuation to induce mechanical forces. Using this system, we then showed that SAECs remain viable, proliferate, and form an epithelial cell monolayer across the entire hammock. By further applying mechanical stimulation via magnetic actuation, we observed an increase in proliferation and strengthening of the cytoskeleton, suggesting an increase in mechanosensing.

Conclusion

This hammock strategy provides an easily accessible and tunable cell culture platform for mimicking distal lung mechanical forces in vitro. We anticipate the promise of this culture platform for mechanistic studies, multi-modal stimulation, and drug or small molecule testing, extendable to other cell types and organ systems.

Introduction

Metastasis is responsible for 90% of cancer-related deaths worldwide. However, the potential inhibitory effects of metastasis by various anticancer drugs have been left largely unexplored. Existing preclinical models primarily focus on antiproliferative agents on the primary tumor to halt the cancer growth but not in metastasis. Unlike primary tumors, metastasis requires cancer cells to exert sufficient cellular traction force through the actomyosin machinery to migrate away from the primary tumor site. Therefore, we seek to explore the potential of cellular traction force as a novel readout for screening drugs that target cancer metastasis.

Methods

In vitro models of invasive and non-invasive breast cancer were first established using MDA-MB-231 and MCF-7 cell lines, respectively. Cellular morphology was characterized, revealing spindle-like morphology in MDA-MB-231 and spherical morphology in MCF-7 cells. The baseline cellular traction force was quantified using the Traction force Microscopy technique. Cisplatin, a paradigm antimetastatic drug, and 5-Fluorouracil (5FU), a non-antimetastatic drug, were selected to evaluate the potential of cellular traction force as a drug testing readout for the in vitro cancer metastasis.

Results

MDA-MB-231 cells exhibited significantly higher baseline cellular traction force compared to MCF-7 cells. Treatment with Cisplatin, an antimetastatic drug, and 5-Fluorouracil (5FU), a non-antimetastatic drug, demonstrated distinct effects on cellular traction force in MDA-MB-231 but not in MCF-7 cells. These findings correlate with the invasive potential observed in the two models.

Conclusion

Cellular traction force emerges as a promising metric for evaluating drug efficacy in inhibiting cancer metastasis using in vitro models. This approach could enhance the screening and development of novel anti-metastatic therapies, addressing a critical gap in current anticancer drug research.