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Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources.

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Blood, tissue and cell establishments (BTCs) stand out in the management of donor selection, procurement and processing of all types of substances of human origin (SoHO). In the last decades, the framework created around BTCs, including hospitals and national health system networks, and their links to research, development and innovation organizations and agencies have spurred their involvement in the study of groundbreaking advanced therapy medicinal products (ATMP). To further improve strategic synergies in the development of ATMPs, it will be required to promote intra- and inter-European collaborations by creating an international network involving BTCs and major stakeholders (i.e., research organizations, hospitals, universities, patient associations, public agencies). This vision is already shared with the European Blood Alliance, the association of non-profit blood establishments, with 26 member states throughout the European Union and European Free Trade Association states. Herein we present and analyze the "BTC for ATMP Development And Manufacture" (BADAM) model, an ethically responsible business model based on the values and missions of BTCs and their commitment to health equity, patient access and education (based on voluntary donation of SoHO to address unmet clinical needs, while contributing to training professionals and scientific literacy of our Society).

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BACKGROUND AIMS: The current approach for preventing hemolysis of red blood cells (RBCs) in major ABO-incompatible bone marrow (BM) grafts after infusion is to deplete RBCs from BM products before transplantation. Traditionally, manual density separation (MDS) using Ficoll-Hypaque (Cytiva Sweden AB, Uppsala, Sweden has been used to accomplish RBC depletion. This process yields good CD34+ cell recovery, but it requires open manipulation and is labor-intensive and time-consuming. We hypothesized that an alternative automated method using Haemonetics Cell Saver 5+ (Haemonetics Corporation, Boston, MA, USA) would offer equivalent RBC depletion and CD34+ cell recovery. Small marrow volumes from pediatric donors can be processed using Cell Saver (CS) without adding the third-party RBCs necessary for other automated methods. METHODS: This retrospective analysis comprised data from 58 allogeneic BM grafts. RBC depletion and CD34+ cell recovery from BM using MDS (35 grafts) were compared with CS (14 grafts). Nine products underwent RBC depletion using CS with Ficoll (CS-F) when RBC volume was less than 125 mL. RESULTS: Linear regression analysis of log transformation of CD34+ cell recovery adjusted for log transformation of both baseline CD34+ cell content and baseline total volume showed no significant difference between MDS and CS (estimated coefficient, -0.121, P = 0.096). All products contained an RBC volume of less than 0.25 mL/kg post-processing. CD34+ cell recovery with CS-F was comparable to MDS and CS and suitable for pediatric recipients of allogeneic hematopoietic cell transplantation. CONCLUSIONS: We provide evidence that an automated method using Haemonetics Cell Saver 5+ achieves RBC depletion and CD34+ cell recovery comparable to MDS when adjusting for baseline factors.

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Cell counting is a fundamental measurement for determining viable cell numbers in biomanufacturing processes. The properties of different cell types and the range of intended uses for cell counts within a biomanufacturing process can lead to challenges in identifying suitable counting methods for each potential application. This is further amplified by user subjectivity in identifying the cells of interest and further identifying viable cells. Replacement of traditionally used manual counting methods with automated systems has alleviated some of these issues. However, a single cell type can exhibit different physical properties at various stages of cell processing which is further compounded by process impurities such as cell debris or magnetic beads. These factors make it challenging to develop a robust cell counting method that offers a high level of confidence in the results. Several initiatives from standards development organizations have attempted to address this critical need for standardization in cell counting. This study utilizes flow-based and image-based methods for the quantitative measurement of cell concentration and viability in the absence of a reference material, based on the tools and guidance provided by the International of Standards (ISO) and the US National Institute of Standards and Technology (NIST). Primary cells were examined at different stages of cell processing in a cell therapy workflow. Results from this study define a systematic approach that enables the identification of counting methods and parameters that are best suited for specific cell types and workflows to ensure accuracy and consistency. Cell counting is a foundational method used extensively along various steps of cell and gene therapy. The standard used in this study may be applied to other cell and gene therapy processes to enable accurate measurement of parameters required to guide critical decisions throughout the development and production process. Using a framework that confirms the suitability of the cell counting method used can minimize variability in the process and final product.

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Higher doses of infused nucleated cells (NCs) are associated with improved clinical outcomes in bone marrow transplantation (BMT) recipients. Most clinicians recommend infusing at least 2.0 × 108 NCs/kg. BMT clinicians request a target NC dose, but the harvested NC dose may be below the requested NC dose even before cell processing. We conducted this retrospective study to investigate the quality of bone marrow (BM) harvest and factors that influence infused NC doses at our institution. We also correlated infused NC doses with clinical outcomes. The study population included 347 BMT recipients (median age, 11 years; range, <1 to 75 years) at the University of Minnesota between 2009 and 2019. Underlying diagnoses mainly included 39% malignant and 61% nonmalignant diagnoses. Requested, harvested, and infused NC doses, as well as cell processing data, were obtained from the Cell Therapy Laboratory; clinical outcomes data were obtained from the University of Minnesota BMT Database. BM harvests were facilitated either by our institution (61%) or by the National Marrow Donor Program (39%). Associations of infused doses with baseline characteristics were assessed using the general Wilcoxon test/Pearson's correlation coefficient. The association of infused dose with neutrophil engraftment (absolute neutrophil count >500) by day 42, platelet engraftment (>20,000) by 6 months, acute graft-versus-host disease grade II-IV, and overall survival (OS) at 5 years were evaluated using regression and Kaplan-Meier curves. The median requested NC dose was 3.0 × 108/kg (range, 2 to 8 × 108/kg), and the median harvested and infused NC doses were 4.0 × 108/kg and 3.6 × 108/kg, respectively. Only 7% of donors had a harvested dose below the minimum requested dose. Moreover, the correlation between requested doses and harvested doses was adequate, with a harvested/requested dose ratio <.5 observed in only 5% of harvests. Additionally, the harvest volume and cell processing method were significantly correlated with the infused dose. Harvest volume exceeding the median of 948 mL was related to a significantly lower infused dose (P < .01). Moreover, hydroxyethyl starch (HES)/buffy coat processing (used to reduce RBCs with major ABO incompatibility) led to a significantly lower infused dose (P < .01). Donor age (median, 19 years; range, <1 to-70 years) and sex did not significantly influence the infused dose. Finally, the infused dose was significantly correlated with neutrophil and platelet engraftment (P < .05) but not with 5-year OS (P = .87) or aGVHD (P = .33). In our program's experience, BM harvesting is efficient and meets the requested minimum dose for 93% of recipients. Harvest volume and cell process play significant roles in determining the final infused dose. Minimizing harvest volume and cell processing could lead to increased infused dose and thus improved outcomes. Moreover, a higher infused dose leads to a better rate of neutrophil and platelet engraftment but not to improved OS, which may be linked to the sample size of our study.

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Introduction: Cell processing operators (CPOs) use a variety of disinfectants that vaporize in the workspace environment. These disinfectants can induce allergic reactions in CPOs, due to their long working hours at cell processing facilities (CPFs). Ionic substances such as CH3COO- generated from peracetic acid, nitrogen oxides (NOx) and sulfur oxides (SOx) from outdoor environment are also known to pollute air. Therefore, our objective was to assess the air quality in CPFs and detect volatile organic compounds (VOCs) from disinfectants and building materials, and airborne ionic substances from outdoor air. Methods: Sampling was conducted at three CPFs: two located in medical institutions and one located at a different institution. Air samples were collected using a flow pump. Ion chromatographic analysis of the anionic and cationic compounds was performed. For VOC analysis, a thermal desorption analyzer coupled with capillary gas chromatograph and flame ionization detector was used. Results: Analysis of the ionic substances showed that Cl-, NOx, and SOx, which were detected in large amounts in the outdoor air, were relatively less in the CPFs. Ethanol was detected as the main component in the VOC analysis. Toluene was detected at all sampling points. As compared to the other environments, air in the incubator contained larger amounts of VOCs, that included siloxane, tetradecane, and aromatics. Conclusions: No VOCs or ionic substances of immediate concern to the health of the CPOs were detected during the non-operating period. However, new clinical trials of cell products are currently underway in Japan, and a variety of new cell products are expected to be approved. With an increase in cell processing, health risks to CPOs that have not been considered previously, may become apparent. We should continue to prepare for the future expansion of the industry using a scientific approach to collect various pieces of information and make it publicly available to build a database.

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The Advanced Therapies Week 2023 conference took place in Miami, FL, USA. Over the course of 4 days packed with talks, panels, company showcases and networking events, a clear message emerged: the future of cell therapy is here. Timely topics covered by speakers and panelists from industry and academia included allogeneic and autologous cell therapies, cell manufacture automation, cell and gene therapy for autoimmune diseases, gene delivery technology, chimeric antigen receptor T-cell therapy in oncology, closed cell therapy manufacturing and how to serve small patient populations. While some challenges remain, this decade will likely witness the US FDA approval of many cell and gene therapies, as well as new devices for their manufacture.

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Several experimental protocols are available to study the synthesis and secretion of proteoglycans in health and diseases, but there are few methods to analyse the intracellular processing of these macromolecules. We report a western blot analysis on medium and cell layer of primary chondrocyte culture to determine the glycanation status of aggrecan. Using a specific antibody against the aggrecan core protein and digesting an aliquot of sample with chondroitinase ABC, it is possible to analyse the whole aggrecan macromolecule and the core protein in order to evaluate defects in aggrecan glycanation.

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Introduction: We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period. Methods: To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase. Results: We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing. Conclusions: Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries.

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Introduction: Cell processing operators (CPOs) are one of the most important human assets for the industrial development of regenerative therapy since cell processing is still carried out manually. However, investigating the psychological aspects of CPOs to take advantage of this important human asset has not been done. Thus, the objectives of the current study were, first, to investigate the attributes of CPOs, second, to identify the factors that give CPOs mental stress, and third, to identify the motivational factors of CPOs. Methods: An online questionnaire was conducted to investigate the attributes of participants, stresses, and motivations; the questionnaire included multiple-choice questions. A survey questionnaire was distributed to 64 operators in 31 CPFs, including universities, clinics, and pharmaceutical companies, from December 2021 to February 2022. Participants responded to the online survey via Microsoft Forms. Results: Approximately half of the CPOs had been employed for two years or less, and they were more likely to experience various stressful situations than CPOs with three or more years of experience. In particular, the wear of dust-free clothing and work in a unique environment of a clean room, where it is difficult to take breaks due to the nature of the closed space, were considered to be particularly stressful situations. However, these CPOs have high motivations based on their pride in being involved in cutting-edge medical care, which is highly expected by society, and in contributing to patient care. Conclusions: While stress with training and education programs for CPOs is an issue that can be resolved through in-house training, other issues that are difficult to resolve immediately, such as mechanization to relieve the stress of human manual operations, were identified. Continued efforts to analyze and alleviate the stress factors identified in this study are expected to improve the quality of CPOs' work and maintain the important human asset of CPOs.

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Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

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Recent developments for the manufacturing of cell-based products have focused on the advancement of products to clinical trials or commercialization, with awareness of the importance of cost-based effectiveness in cell manufacturing. The mechanization of cell-processing operations is advantageous for the reproducibility and stability of product quality and is thought to reduce the cost-of-goods through the life cycle of the product in a scale-up system; however, few cases of the implementation exist. This study developed an estimation method for the resource expenditure of cell-processing operations in the manufacturing of cell-based products. To estimate resource expenditures, we evaluated the manufacturing processes by operations involving entering into the surrounding area of cell processing zone, materials loading, cell-processing operation, cleaning, and leaving from the surrounding area. The cell-processing operation is applicable to manual or robotic cell manufacturing system in a biosafety cabinet or an isolator system. In cases of low annual batch numbers of manufacturing (batch number <33), the resource expenditure of cell-processing operations in a robotic operation system installed in the isolator system is estimated to be higher compared with a manual operation system in the isolator system due to additional initial costs for design and fabrication of the robotic operation system containing robot arms. With increasing numbers of annual batches, the resource expenditure decreases for robotic operating system, leading to an advantageous juncture where the resource expenditure of a robotic operation system is equivalent to that of a manually operated system, whereby the labor cost for cell-processing operations rises. In addition, the expertise of operations required for cell manufacturing is suggested to foster potential risks associated with the operation skills or turnover of operators, and the cost of education and training increases due to the necessity of persistent human resource development. Collectively, revealing the approach for installation of robotic operation system in cell manufacturing.

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Before the viability of a cell formulation can be assessed for implementation in commercial sodium ion batteries, processes applied in cell production should be validated and optimized. This review summarizes the steps performed in constructing sodium ion (Na-ion) cells at research scale, highlighting parameters and techniques that are likely to impact measured cycling performance. Consistent process-structure-performance links have been established for typical lithium-ion (Li-ion) cells, which can guide hypotheses to test in Na-ion cells. Liquid electrolyte viscosity, sequence of mixing electrode slurries, rate of drying electrodes and cycling characteristics of formation were found critical to the reported capacity of laboratory cells. Based on the observed importance of processing to battery performance outcomes, the current focus on novel materials in Na-ion research should be balanced with deeper investigation into mechanistic changes of cell components during and after production, to better inform future designs of these promising batteries.

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Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation-based model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 106  cell h-1 ) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.

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BACKGROUND: The complex isolation and purification process of hepatocytes for transplantation is labor intensive and with great contamination risk. Here, as a pilot and feasibility study, we examined in vitro and in vivo hepatocyte isolation feasibility and cell function of Cell Saver® Elite®, an intraoperative blood-cell-recovery system. METHODS: Rat and pig liver cells were collected using this system and then cultured in vitro, and their hepatocyte-specific enzymes were characterized. We then transplanted the hepatocytes in an established acute liver-injured (retrorsine+D-galactosamine-treated) rat model for engraftment. Recipient rats were sacrificed 1, 2, and 4 weeks after transplantation, followed by donor-cell identification and histological, serologic, and immunohistopathological examination. To demonstrate this Cell Saver® strategy is workable in the first place, traditional (classical) strategy, in our study, behaved as certainty during the cell manufacturing process for monitoring quality assurance throughout the course, from the start of cell isolation to post-transplantation. RESULTS: We noted that in situ collagenase perfusion was followed by filtration, centrifugation, and collection in the Cell Saver® until the process ended. Most (>85%) isolated cells were hepatocytes (>80% viability) freshly demonstrating hepatocyte nuclear factor 4α and carbamoyl-phosphate synthase 1 (a key enzyme in the urea cycle), and proliferating through intercellular contact in culture, with expression of albumin and CYP3A4. After hepatocyte transplantation in dipeptidyl peptidase IV (-/-) rat liver, wild-type donor hepatocytes engrafted and repopulated progressively in 4 weeks with liver functional improvement. Proliferating donor hepatocyte-native biliary ductular cell interaction was identified. Post-transplantation global liver functional recovery after Cell Saver and traditional methods was comparable. CONCLUSIONS: Cell Saver® requires reduced manual manipulation for isolating transplantable hepatocytes.

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INTRODUCTION: We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF). METHODS: Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated. RESULTS: The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials. CONCLUSIONS: The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

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Cell processing laboratories are an important part of cancer treatment centers. Cell processing laboratories began by supporting hematopoietic stem cell (HSC) transplantation programs. These laboratories adapted closed bag systems, centrifuges, sterile connecting devices and other equipment used in transfusion services/blood banks to remove red blood cells and plasma from marrow and peripheral blood stem cells products. The success of cellular cancer immunotherapies such as Chimeric Antigen Receptor (CAR) T-cells has increased the importance of cell processing laboratories. Since many of the diseases successfully treated by CAR T-cell therapy are also treated by HSC transplantation and since HSC transplantation teams are well suited to manage patients treated with CAR T-cells, many cell processing laboratories have begun to produce CAR T-cells. The methods that have been used to process HSCs have been modified for T-cell enrichment, culture, stimulation, transduction and expansion for CAR T-cell production. While processing laboratories are well suited to manufacture CAR T-cells and other cellular therapies, producing these therapies is challenging. The manufacture of cellular therapies requires specialized facilities which are costly to build and maintain. The supplies and reagents, especially vectors, can also be expensive. Finally, highly skilled staff are required. The use of automated equipment for cell production may reduce labor requirements and the cost of facilities. The steps used to produce CAR T-cells are reviewed, as well as various strategies for establishing a laboratory to manufacture these cells.

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``Cellular therapies first emerged as specialized therapies only available at a few "boutique" centers worldwide. To ensure broad access to these investigational therapies-regardless of geography, demographics and other factors-more and more academic clinical trials are becoming multi-center. Such trials are typically performed with a centralized manufacturing facility receiving the starting material and shipping the final product, either fresh or cryopreserved, to the patient's institution for infusion. As these academic multi-center trials increase in number, it is critical to have procedures and training programs in place to allow these sites that are remote from the production facility to successfully participate in these trials and satisfy regulatory compliance and patient safety best practices. Based on the collective experience of the Consortium for Pediatric Cellular Immunotherapy, the authors summarize the challenges encountered by institutions in shipping and receiving the starting material and final product as well as preparing the final product for infusion. The authors also discuss best practices implemented by each of the consortia institutions to overcome these challenges.

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Current therapies for novel coronavirus disease (COVID-19) are generally used to manage rather than cure this highly infective disease. Therefore, there is a significant unmet medical need for a safe and effective treatment for COVID-19. Inflammation is the driving force behind coronavirus infections, and the majority of deaths caused by COVID-19 are the result of acute respiratory distress syndrome (ARDS). It is crucial to control the inflammation as early as possible. To date, numerous studies have been conducted to evaluate the safety and efficacy of tissue engineering and regenerative medicine (TERM) products, including mesenchymal stem cells (MSCs), and their derivatives (eg, exosomes) for coronavirus infections, which could be applied for the COVID-19. In this review, first, the impacts of the COVID-19 pandemic in the present and future of TERM research and products are briefly presented. Then, the recent clinical trials and the therapeutic benefits of MSCs in coronavirus-induced ARDS are critically reviewed. Last, recent advances in the field of tissue engineering relevant to coronavirus infections, including three-dimensional platforms to study the disease progression and test the effects of antiviral agents, are described. Moreover, the application of biomaterials for vaccine technology and drug delivery are highlighted. Despite promising results in the preclinical and clinical applications of MSC therapy for coronavirus infections, controversy still exists, and thus further investigation is required to understand the efficacy of these therapies.

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Peptide subunit vaccines increase safety by reducing the risk of off-target responses and improving the specificity of the induced adaptive immune response. The immunogenicity of most soluble peptides, however, is often insufficient to produce robust and lasting immunity. Many biomaterials and delivery vehicles have been developed for peptide antigens to improve immune response while maintaining specificity. Peptide nanoclusters (PNC) are a subunit peptide vaccine material that has shown potential to increase immunogenicity of peptide antigens. PNC are comprised only of crosslinked peptide antigen and have been synthesized from several peptide antigens as small as 8 amino acids in length. However, as with many peptide vaccine biomaterials, synthesis requires adding residues to the peptide and/or engaging amino acids within the antigen epitope covalently to form a stable material. The impact of antigen modifications made to enable biomaterial incorporation or formation is rarely investigated, since the goal of most studies is to compare the soluble antigen with biomaterial form of antigen. This study investigates PNC as a platform vaccine biomaterial to evaluate how peptide modification and biomaterial formation with different crosslinking chemistries affect epitope-specific immune cell presentation and activation. Several types of PNC were synthesized by desolvation from the model peptide epitope SIINFEKL, which is derived from the immunogenic protein ovalbumin. SIINFEKL was altered to include extra residues on each end, strategically chosen to enable multiple conjugation chemistry options for incorporation into PNC. Several crosslinking methods were used to control which functional groups were used to stabilize the PNC, as well as the reducibility of the crosslinking. These variations were evaluated for immune responses and biodistribution following in vivo immunization. All modified antigen formulations still induced comparable immune responses when incorporated into PNC compared to unmodified soluble antigen alone. However, some crosslinking methods led to a significant increase in desirable immune responses while others did not, suggesting that not all PNC were processed the same. These results help guide future peptide vaccine biomaterial design, including PNC and a wide variety of conjugated and self-assembled peptide antigen materials, to maximize and tune the desired immune response.

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