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Abstract Hepatic sinusoids play a key role in maintaining high activities of liver cells in the hepatic acinus. However, the construction of hepatic sinusoids has always been a challenge for liver chips, especially for large-scale liver microsystems. Herein, we report an approach for the construction of hepatic sinusoids. In this approach, hepatic sinusoids are formed by demolding a self-developed microneedle array from a photocurable cell-loaded matrix in a large-scale liver-acinus-chip microsystem with a designed dual blood supply. Primary sinusoids formed by demolded microneedles and spontaneously self-organized secondary sinusoids can be clearly observed. Benefiting from significantly enhanced interstitial flows by formed hepatic sinusoids, cell viability is witnessed to be considerably high, liver microstructure formation occurs, and hepatocyte metabolism is enhanced. In addition, this study preliminarily demonstrates the effects of the resulting oxygen and glucose gradients on hepatocyte functions and the application of the chip in drug testing. This work paves the way for the biofabrication of fully functionalized large-scale liver bioreactors.

As a foundation of life-science research and advancement, biobanking has played a critical role and made tremendous contributions to healthcare, biotechnology, disease control and prevention, as well as bio-conservation for the benefit of all humankind. This paper starts with a brief introduction of basic concepts, history, classification, and significance of biobanking, followed by a discussion on cryobiology fundamentals and key challenges faced by cryopreservation in biobanking. A special case discussion on the cryopreservation and biobanking of pathogenic microorganisms to meet both the unmet needs for biomedical research and the urgent demand for developing countermeasures against the ongoing coronavirus disease 2019 (COVID-19) pandemic is highlighted with insightful recommendations for future studies.

IntroductionFine-tuning (FT) is a generally adopted transfer learning method for deep learning-based magnetic resonance imaging (MRI) reconstruction. In this approach, the reconstruction model is initialized with pre-trained weights derived from a source domain with ample data and subsequently updated with limited data from the target domain. However, the direct full-weight update strategy can pose the risk of "catastrophic forgetting" and overfitting, hindering its effectiveness. The goal of this study is to develop a zero-weight update transfer strategy to preserve pre-trained generic knowledge and reduce overfitting.MethodsBased on the commonality between the source and target domains, we assume a linear transformation relationship of the optimal model weights from the source domain to the target domain. Accordingly, we propose a novel transfer strategy, linear fine-tuning (LFT), which introduces scaling and shifting (SS) factors into the pre-trained model. In contrast to FT, LFT only updates SS factors in the transfer phase, while the pre-trained weights remain fixed.ResultsTo evaluate the proposed LFT, we designed three different transfer scenarios and conducted a comparative analysis of FT, LFT, and other methods at various sampling rates and data volumes. In the transfer scenario between different contrasts, LFT outperforms typical transfer strategies at various sampling rates and considerably reduces artifacts on reconstructed images. In transfer scenarios between different slice directions or anatomical structures, LFT surpasses the FT method, particularly when the target domain contains a decreasing number of training images, with a maximum improvement of up to 2.06 dB (5.89%) in peak signal-to-noise ratio.DiscussionThe LFT strategy shows great potential to address the issues of "catastrophic forgetting" and overfitting in transfer scenarios for MRI reconstruction, while reducing the reliance on the amount of data in the target domain. Linear fine-tuning is expected to shorten the development cycle of reconstruction models for adapting complicated clinical scenarios, thereby enhancing the clinical applicability of deep MRI reconstruction.

Magnetic resonance (MR) images from low-field scanners present poorer signal-to-noise ratios (SNRs) than those from high-field scanners at the same spatial resolution. To obtain a clinically acceptable SNR, radiologists operating the low-field scanners use a much smaller acquisition matrix than high-field scanners. Thus, the current state of the image quality indicates the need for further research to improve the image quality of low-field systems. Strategies based on super-resolution (SR) techniques can be alternatives for image reconstruction. However, predetermined degradation methods embedded in these techniques, such as bicubic downsampling, seem to impose a performance drop when the actual degradation is different from the pre-defined assumption. In this study, we collected a unique dataset by scanning 70 participants to address this problem. The anatomical locations of the scanned image slices were the same for 0.35T and 3T data. Low-resolution (LR) images (0.35T) and high-resolution (HR) images (3T) were the image pairs used for data training. Herein, we introduce a novel CNN-based network with hybrid attention mechanisms (HybridAttentionResNet, HARN) to adaptively capture diverse information and reconstruct super-resolution 0.35T MR images (3T-like MR images). Specifically, the proposed dense block combines variant dense blocks and attention blocks to extract abundant features from LR images. The experimental results demonstrate that our proposed residual network efficiently recovers significant textures while rendering a high peak signal-to-noise ratio (PSNR) and an appealing structural similarity index (SSIM). Moreover, an extensive subjective-mean-opinion-score (SMOS) proves to be promising in the clinical application using HARN.

BackgroundHemoglobin A1c (HbA1c) variability may be a predictor of diabetic complications, but the predictive values of HbA1c trajectories remain unclear. We aimed to classify long-term HbA1c trajectories and to explore their effects on future clinical outcomes in a 10-year cohort with type 2 diabetes mellitus (T2DM).MethodsA total of 2,161 participants with T2DM from the Beijing Community Diabetes Study were included. The 10-year follow-up was divided into two stages for the present data analysis. Stage I (from 2008 to 2014) was used to identify the HbA1c trajectories and to calculate the adjusted SD of HbA1c (HbA1c-adjSD), or the coefficient of variation of HbA1c (HbA1c-CV). Stage II (from 2014 to 2018) was used to collect the records of the new occurrence of diabetes-related clinical outcomes. Latent growth mixture models were used to identify HbA1c trajectories. Cox proportional hazards models were used to explore the relationship between HbA1c trajectories, HbA1c-adjSD, or HbA1c-CV and the future outcomes.ResultsThree HbA1c trajectories were identified, including low stable (88.34%), gradual decreasing (5.83%), and pre-stable and post-increase (5.83%). Either the risk of death or the chronic complications were significantly higher in the latter two groups compared to the low stable group after adjustment for average HbA1c and other traditional risk factors, the adjusted hazard ratios (HRs) for renal events, composite endpoint, and all-cause death for the pre-stable and post-increase group were 2.83 [95%CI: 1.25–6.41, p = 0.013], 1.85 (95%CI: 1.10–3.10, p = 0.020), and 3.01 (95%CI: 1.13–8.07, p = 0.028), respectively, and the adjusted HR for renal events for the gradual decreasing group was 2.37 (95%CI: 1.08–5.21, p = 0.032). In addition, both univariate and multivariate Cox HR models indicated that participants in the fourth and third quartiles of HbA1c-CV or HbA1c-adjSD were at higher risk of renal events compared to participants in the first quartile.ConclusionsHbA1c trajectories, HbA1c-CV, and HbA1c-adjSD could all predict diabetes-related clinical outcomes. HbA1c trajectories could reflect long-term blood glucose fluctuation more intuitively, and non-stable HbA1c trajectories may predict increased risk of renal events, all-cause death, and composite endpoint events, independent of average HbA1c.

BackgroundCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.Methods and findingsTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.ConclusionsCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.

BACKGROUND:Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. METHODS AND FINDINGS:To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. CONCLUSIONS:Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.

An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6)-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p

The Sf21 cell line is extensively used for virus research and producing heterologous recombinant proteins. To develop optimal strategies for minimizing cell injury due to intracellular ice formation and excessive volume shrinkage during cryopreservation, the fundamental transport properties including the osmotic inactive volume (Vb ), the hydraulic conductivity (Lp ), and the glycerol permeability (Ps ) of Sf21 cell membrane at 25, 15, 5 and -2°C were characterized using a micro-perfusion chamber. The effects of temperature on the hydraulic conductivity and the glycerol permeability of Sf21 cell membrane, reflected by the activation energies, were quantitatively investigated. It was found that the hydraulic conductivity decreases along with the increase of the final CPA concentration at a given temperature, and quantitative analysis indicates that the hydraulic conductivity has a significant linear attenuation along with the increase of the concentration of glycerol. Therefore, we incorporate the concentration dependence of the hydraulic conductivity into the classic Arrhenius relationship by replacing the constant reference value of the hydraulic conductivity at the reference temperature with a function that is linearly dependent on the CPA concentration. Consequently, the prediction of the Arrhenius relationship is improved, and the novel Arrhenius relationship could be very important to the development of optimal strategies for cell cryopreservation.

A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypoosmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm/kg, exocrine cell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet cells lost membrane integrity and mitochondrial function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.