Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Alginate Affects Bioactivity of Chimeric Collagen-Binding LL37 Antimicrobial Peptides Adsorbed to Collagen-Alginate Wound Dressings.

Chronic infected wounds cause more than 23,000 deaths annually. Antibiotics and antiseptics are conventionally used to treat infected wounds; however, they can be toxic to mammalian cells, and their use can contribute to antimicrobial resistance. Antimicrobial peptides (AMPs) have been utilized to address the limitations of antiseptics and antibiotics. In previous work, we modified the human AMP LL37 with collagen-binding domains from collagenase (cCBD) or fibronectin (fCBD) to facilitate peptide tethering and delivery from collagen-based wound dressings. We found that cCBD-LL37 and fCBD-LL37 were retained and active when bound to 100% collagen scaffolds. Collagen wound dressings are commonly made as composites with other materials, such as alginate. The goal of this study was to investigate how the presence of alginate affects the tethering, release, and antimicrobial activity of LL37 and CBD-LL37 peptides adsorbed to commercially available collagen-alginate wound dressings (FIBRACOL Plus-a 90% collagen and 10% alginate wound dressing). We found that over 85% of the LL37, cCBD-LL37, and fCBD-LL37 was retained on FIBRACOL Plus over a 14-day release study (90.3, 85.8, and 98.6%, respectively). Additionally, FIBRACOL Plus samples loaded with peptides were bactericidal toward Pseudomonas aeruginosa, even after 14 days in release buffer but demonstrated no antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. The presence of alginate in solution induced conformational changes in the cCBD-LL37 and LL37 peptides, resulting in increased peptide helicity, and reduced antimicrobial activity against P. aeruginosa. Peptide-loaded FIBRACOL Plus scaffolds were not cytotoxic to human dermal fibroblasts. This study demonstrates that CBD-mediated LL37 tethering is a viable strategy to reduce LL37 toxicity, and how substrate composition plays a crucial role in modulating the antimicrobial activity of tethered AMPs.

A Method for High-Throughput Robotic Assembly of Three-Dimensional Vascular Tissue.

IMPACT STATEMENT: Self-assembled tissues have potential to serve both as implantable grafts and as tools for disease modeling and drug screening. For these applications, tissue production must ultimately be scaled-up and automated. Limited technologies exist for precisely manipulating self-assembled tissues, which are fragile early in culture. Here, we presented a method for automatically stacking self-assembled smooth muscle cell rings onto mandrels, using a custom-designed well plate and robotic punch system. Rings then fuse into tissue-engineered blood vessels (TEBVs). This is a critical step toward automating TEBV production that may be applied to other tubular tissues as well.

irRECIST for the Evaluation of Candidate Biomarkers of Response to Nivolumab in Metastatic Clear Cell Renal Cell Carcinoma: Analysis of a Phase II Prospective Clinical Trial.

PURPOSE: Immune-related RECIST (irRECIST) were designed to capture atypical responses seen with immunotherapy. We hypothesized that, in patients with metastatic clear cell renal cell carcinoma (mccRCC), candidate biomarkers for nivolumab response would show improved association with clinical endpoints capturing atypical responders (irRECIST) compared with standard clinical endpoints (RECISTv1.1). EXPERIMENTAL DESIGN: Endpoints based on RECISTv1.1 [objective response rate (ORR)/progression-free survival (PFS)] or irRECIST [immune-related ORR (irORR)/immune-related PFS (irPFS)] were compared in patients enrolled in the CheckMate-010 trial. Pretreatment tumors were analyzed by PD-L1 and PD-L2 IHC, and by multiplex immunofluorescence for CD8, PD-1, TIM-3, and LAG-3. T-cell activation signatures were assessed by RNA sequencing. RESULTS: Median irPFS was significantly longer than median PFS. irORR was not significantly different from ORR, but immune-related progressive disease (irPD) rate was significantly lower than progressive disease (PD) rate. Tumor cell (TC) PD-L1 expression was not associated with PFS or ORR, but patients with TC PD-L1 ≥1% had longer median irPFS and higher irORR. High percentage of CD8+ tumor-infiltrating cells (TIC) that are PD-1+TIM-3-LAG-3- (% CD8+PD-1+TIM-3-LAG-3- TIC) correlated with high levels of T-cell activation and was associated with longer median irPFS and higher irORR. Notably, combination of TC PD-L1 expression with % CD8+PD-1+TIM-3-LAG-3- TIC identified three groups of patients for which irPFS and irORR were significantly different. CONCLUSIONS: Atypical responders to nivolumab were identified in the CheckMate-010 trial. We observed improved association of candidate biomarkers for nivolumab response with endpoints defined by irRECIST compared with RECISTv1.1. TC PD-L1 expression in combination with PD-1 expression on CD8+ TIC may predict outcome on nivolumab in mccRCC.

Collagen tethering of synthetic human antimicrobial peptides cathelicidin LL37 and its effects on antimicrobial activity and cytotoxicity.

Wound infections, particularly of chronic wounds, pose a substantial challenge for designing antimicrobial dressings that are both effective against pathogens, and do not interfere with wound healing. Due to their broad-spectrum antimicrobial and immunomodulatory activities, naturally-occurring antimicrobial peptides (AMPs) are promising alternative treatments. However, their cytotoxicity at high concentrations and poor stability hinders their clinical use. To mitigate these undesirable properties, we investigated the effects of tethering human AMP cathelicidin LL37 to collagen, one of the main extracellular matrix proteins in wound sites, secreted by fibroblasts, and in commercially-available wound dressings. The active domain of human AMP cathelicidin, LL37, and two chimeric peptides containing LL37 fused to collagen binding domains (derived from collagenase - cCBD-LL37 or fibronectin - fCBD-LL37) were synthesized and adsorbed to PURACOL® type I collagen scaffolds. After 14days, 73%, 81% and 99% of LL37, cCBD-LL37 and fCBD-LL37, respectively, was retained on the scaffolds and demonstrated undiminished antimicrobial activity when challenged with both Gram-positive and Gram-negative bacterial strains. Loaded scaffolds were not cytotoxic to fibroblasts despite retaining peptides at concentrations 24 times higher than the reported cytotoxic concentrations in solution. These findings indicate that biopolymer-tethered AMPs may represent a viable alternative for preventing and treating wound infection while also supporting tissue repair. STATEMENT OF SIGNIFICANCE: Over 6.5million people annually in the United States suffer chronic wounds; many will become infected with antibiotic-resistant bacteria. Treatments used to prevent and fight infection are toxic and may hinder wound healing. AMPs are broad-spectrum antimicrobials that also promote healing; however, their instability and toxicity are major challenges. To overcome treatment gaps, we functionalized collagen scaffolds with chimeric antimicrobial peptides (AMPs) with collagen binding domains to create antimicrobial and non-cytotoxic scaffolds that may promote healing. This is the first report of CBD-mediated delivery of AMPs onto collagen scaffolds that demonstrates no cytotoxicity toward fibroblasts. This study also suggests that retention of antimicrobial activity is CBD-dependent, which provides foundations for fundamental studies of CBD-AMP mechanisms and clinical explorations.