John Bischof’s research while affiliated with University of Minnesota and other places

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Publications (38)


Fig. 3. Viability and tissue morphology directly post cryopreservation. (A) (i-v) AO/PI staining where AO-stained cells appear green, and the PI-stained cells are red, indicating cell membrane compromise. (vi-x) H&E staining of the slice groups just post rewarming. (B) The viability through membrane integrity was assessed by analyzing the number of dead cells in the given z-plane by assessing areas of individual cells and comparing the number of PI quenched cells to the total number of cells. (C) Viability measure from AO/PI for each group. Levels of significance: ***, p =0.0001; ****, p <0.0001 (One-way ANOVA). AO, acridine orange; CPA, cryoprotective agent; FT, slow freezing and rapid thawing; H&E, hematoxylin and eosin; PI, propidium iodide; VR, vitrification and rewarming.
Fig 4. Metabolic and functional assessments of liver slices over 3 days in culture. (A) ATP assessment. (B) Urea production. (c) Albumin synthesis. Levels of significance: *p
Fig. 6. Drug response of PCLS. (A) ATP levels at the end of day 3 in culture of control and VR slices exposed to varying concentrations of Acetaminophen (APAP). (B) Urea levels measured normalized to 0mM concentration spanning a 3-day culture period on exposure to different APAP concentrations. Data are mean ± standard deviation. Levels of significance are represented by compact letter display (Dunn's test). APAP, N-acetyl-para-aminophenol (acetaminophen), VR, vitrification, and rewarming.
Vitrification and rapid rewarming of precision-cut liver slices for pharmacological and biomedical research
  • Preprint
  • File available

December 2024

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28 Reads

Srivasupradha Ramesh

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Bat-Erdene Namsrai

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[...]

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John C. Bischof

Background and Aims: High-throughput in vitro pharmacological toxicity testing is essential for drug discovery. Precision-cut liver slices (PCLS) provide a robust system for screening that is more representative of the complex 3D structure of the whole liver than isolated hepatocytes. However, PCLS are not available as off-the-shelf products, significantly limiting their translational potential. Cryopreservation could solve this bottleneck by effectively preserving PCLS indefinitely until their time of use. Conventional cryopreservation (slow cooling in DMSO-forming ice) results in poor PCLS viability and function and, therefore, has proven unsuitable. Here, we explore an ice-free cryopreservation approach called vitrification and focus on culturing and assessing PCLS for 3 days post-vitrification and rewarming, given that most acute drug toxicity tests are conducted over 24h. Methods: Rat liver slices were diffusively loaded with a cryoprotective agent (CPA) cocktail consisting of EG and Sucrose. The CPA-loaded PCLS were placed on a polymer cryomesh, vitrified in liquid nitrogen (LN2), and rapidly rewarmed in CPA. The vitrified and rewarmed PCLS were subsequently cultured in a controlled volume of serum-free, chemically defined media for 3 days. Results: The cryopreserved PCLS maintained high viability, morphology, function, enzymatic activity, and drug toxicity response. Results show that the vitrified PCLS perform comparably to untreated controls and significantly outperform conventionally cryopreserved PCLS in all assessments (p < 0.05). Conclusions: Rapid vitrification and rewarming of PCLS using cryomesh enabled successful preservation and culture. This approach maintained high viability, function, enzymatic activity, and drug response for 3 days in culture, similar to controls.

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A Model of Cryosurgical Destruction in AT-1 Prostate Tumor Based on Cellular Damage Mechanisms

September 2024

The thermal history during a prostate cryosurgery is known to lead to different cooling rates, end-temperatures and end-times within a cryosurgical iceball. The tissue exposed to this range of thermal histories will experience thermally-induced biophysical events which affect cell viability (dehydration and intracellular ice formation. IIF), injury due solely to the temperature and time of exposure, and injury due to host response. In this study, the dehydration and IIF behavior of single AT-1 prostate cancer cells is experimentally measured, the biophysical parameters of water transport and IIF are obtained, and the probability of injury to single AT-1 cells due to dehydration (Prd) and IIF (PIF) is predicted for a variety of cryosurgically-relevant thermal histories. In addition, viability data obtained after cooling to different end-temperatures at constant coolins rates is used to create an empirical function of injury for single AT-1 cells based solely on end-temperature (Pre). A total cellular injury model which then combines the biophysical mechanisms of injury as well as the empirically-obtained temperature viability function is created. This model is used to predict worst-case (i.e. highest possible survival) cryosurgical destruction in an AT-1 tumor; the implications for clinical cryosurgery are discussed.



Measurement of Water Transport During Freezing Using Differential Scanning Calorimetry

July 2023

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7 Reads

A new technique using Differential Scanning Calorimeter (DSC) was developed to investigate water transport in whole tissue slices (1–5 mm3) and suspended cells during freezing. The tissue and cellular DSC data were correlated to water transport data by freeze substitution tissue microscopy and standard cellular cryomicroscopy techniques respectively. Sprague Dawley liver tissue and a (non-attached) lymphocyte (Epstein Bar Virus Transformed, EBVT) human cell system, were chosen as our tissue and cell model systems. The DSC was used to quantitatively monitor the heat released by water transported from the cell to the frozen vascular/ extracellular space in both systems at 5°C/min. Cryomicroscopy experiments verified that at a slow cooling rate of 5°C/min no intracellular ice formation (IIF) occurred in either system. The sub-zero volumes of the tissue and cells were obtained as a function of temperature by both DSC and cryomicroscopy. By fitting a model of water transport for cells and tissues, dV/dt = f (Vb, B, T (t), Lp (Lpg, ELp)), to the DSC data for both systems, the following biophysical parameters were obtained, for rat liver tissue: Lpg = 2.25 μm/min-atm, ELp = 75.76 kcal/mole, and for EBVT lymphocytes: Lpg = 0.15 μm/min-atm, ELp = 28.78 kcal/mole. These results compare favorably to a recent study which found water transport parameters in whole liver tissue (Pazhayannur and Bischof, 1996) and to the single cell cryomicroscopy data we obtained in this study. The DSC technique is shown to be a fast and powerful method to obtain dynamic water transport information during cell and tissue freezing.


Fig. 5 Comparison between the existing and the optimized VMP loading protocols. a, e The arterial concentration and pressure during perfusion loading. b, f The model-predicted and experimental perfusion resistances for the representative cases. R 2 values equal 0.987 and 0.983 for b and f, respectively. c, g The model-predicted CPA concentration inside the kidney tissue for the representative cases. d,
Fig. 6 Physical (vitrification) and biological assessments of rat kidneys loaded by the two protocols. a, b Representative photo and micro-CT image of vitrified kidney perfused with the existing loading protocol. d, e Representative photo and micro-CT image of vitrified kidney perfused with the optimized loading protocol. Uniform Hounsfield units (HU) ~500 (yellow-orange) in the micro-CT images
Model-Guided Design and Optimization of CPA Perfusion Protocols for Whole Organ Cryopreservation

June 2023

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138 Reads

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7 Citations

Annals of Biomedical Engineering

Vitrification could enable long-term organ preservation, but only after loading high-concentration, potentially toxic cryoprotective agents (CPAs) by perfusion. In this paper, we combine a two-compartment Krogh cylinder model with a toxicity cost function to theoretically optimize the loading of CPA (VMP) in rat kidneys as a model system. First, based on kidney perfusion experiments, we systematically derived the parameters for a CPA transport loading model, including the following: Vb = 86.0% (ra = 3.86 μm), Lp = 1.5 × 10-14 m3/(N·s), ω = 7.0 × 10-13 mol/(N·s), σ = 0.10. Next, we measured the toxicity cost function model parameters as α = 3.12 and β = 9.39 × 10-6. Combining these models, we developed an improved kidney-loading protocol predicted to achieve vitrification while minimizing toxicity. The optimized protocol resulted in shorter exposure (25 min or 18.5% less) than the gold standard kidney-loading protocol for VMP, which had been developed based on decades of empirical practice. After testing both protocols on rat kidneys, we found comparable physical and biological outcomes. While we did not dramatically reduce toxicity, we did reduce the time. As our approach is now validated, it can be used on other organs lacking defined toxicity data to reduce CPA exposure time and provide a rapid path toward developing CPA perfusion protocols for other organs and CPAs.







Citations (13)


... 101 Several studies have demonstrated that the colloidal and thermal stability of MNPs in VS55 can be maintained though surface coating with resorcinol-formaldehyde resin or silica. [104][105][106][107] Additionally, surface modification with poly(ethylene glycol) has been shown to reduce cellular interactions and thus cytotoxicity. 106,107 Manuchehrabadi achieved nanowarming of porcine arteries and porcine aortic heart valve leaflet tissues using magnetic heating. ...

Reference:

Advances in magnetic nanoparticles for molecular medicine
Engineering Magnetic Nanoclusters for Highly Efficient Heating in Radio-Frequency Nanowarming
  • Citing Article
  • April 2024

Nano Letters

... Hence, the minimum CPA concentration for vitrification would be ~62% w/w, which is slightly lower than M22 (~66%w/w which includes carrier solution), where we have shown successful vitrification at 3L. Higher concentrations of CPAs such as VS83 (83% w/w CPA) have even lower CCR and can be more easily vitrified but increase biological toxicity relative to the CPAs chosen here [34]. To remain at a lower concentration of CPA and still achieve vitrification at higher volumes without toxicity, future work can assess the impact of ice recrystallization inhibitors (IRIs), polymers (e.g., polyglycerol-PGL, polyvinyl alcohol-PVA, polyethylene glycol-PEG, x-1000, z-1000, etc.), or other novel cryoprotective agents [35,36]. ...

Model-Guided Design and Optimization of CPA Perfusion Protocols for Whole Organ Cryopreservation

Annals of Biomedical Engineering

... Therefore warming rate is an important consideration in combatting freezing damage (Gao and Critser, 2000;Waters et al., 2020). In particular, a sample that has undergone vitrification may be especially susceptible to ice recrystallization if the warming rate is too slow (Bojic et al., 2021;Zhan et al., 2022). ...

Rapid joule heating improves vitrification based cryopreservation
  • Citing Article
  • December 2022

Cryobiology

... Anti-PD-1 immunotherapy, which has been approved for treating MSI-H and dMMR colorectal cancer, has also been demonstrated to be effective in preclinical colon cancer models including MC-38 15,16 . Immunotherapy was delivered by intraperitoneal injecting 100 μg of antibody (InVivoMAb anti-mouse PD-1, Clone RMP1-14 from Bio X Cell) on days 1, 3, and 5 on the mice bearing MC-38 tumor. ...

Tumor Ablation by Irreversible Electroporation Augments PD-1 Checkpoint Inhibitor Immunotherapy in Colon Adenocarcinoma
  • Citing Article
  • October 2022

Journal of the American College of Surgeons

... Specimens have to be stored below −130 °C to avoid devitrification. Functional cryopreservation by vitrification is an active field of basic research, and has been successfully applied to the rat kidney and liver [31,32]. Functional cryopreservation has not yet been demonstrated for the whole adult mammalian brain, let alone body [33][34][35][36][37][38][39][40]. ...

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming
  • Citing Article
  • October 2022

Annals of Biomedical Engineering

... The successful nanowarming of tissues [196] and small animal organs [157,197] has yielded encouraging results. In a recent study, Han et al. [159] successfully transplanted a nanowarmed, vitrified rat kidney that had been preserved for 100 days. ...

Vitrification and Rewarming of Magnetic Nanoparticle‐Loaded Rat Hearts

... Cell viability was between 90 and 87 %. The works [24,25,26,27] used aluminum foil or block and copper disc with different droplet injection methods, such as acoustic droplet injection, inkjet printing and micropipette and pressure pulse, respectively. Volumes in the order of pL to μL were used, and high cooling rates and cell viabilities above 80 % were found. ...

Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation

... The limitations of CTPA include radiation exposure, potential nephrotoxicity from iodinated contrast, and high cost [21]. The key challenges include distinguishing between true emboli and artifacts and differentiating non-PE findings like the "pulmonary wedge appearance" or an endoluminal ring in the azygous vein [22]. CTPA remains a reliable diagnostic tool, with excellent specificity, sensitivity, and negative predictive value for both massive and non-massive PE. ...

Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis

ACS Nano

... Once the device's reliability was confirmed, we obtained continuous data and meticulously documented it for the purpose of calculating temperature variation per second. This approach enabled us to utilize more precise methodologies, such as the Lucas box method and the increment-corrected method, to derive the SAR value [39,41]. ...

The impact of data selection and fitting on SAR estimation for magnetic nanoparticle heating

... The preservation of the optical properties was the focus due to its direct impact on the thermal performance of the solution. The extinction coefficient of TiN NPs, TiN clusters, and GNRs was evaluated by suspending the nanomaterials in 15% methanol (MeOH), 7.5% propylene glycol (PG), and 20% polyethylene glycol (PEG), a CPA solution developed by Smith et al. (2020) to successfully vitrify a biomaterial microdroplet. Solutions of 5 mL CPAs with concentrations as high as 100 μg/ mL of each nanomaterial were sonicated for 6 min to obtain homogenized samples. ...

High throughput cryopreservation of aquaculture seed
  • Citing Article
  • December 2020

Cryobiology