Michael Etheridge’s research while affiliated with University of Minnesota and other places

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Publications (57)


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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18 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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Physical vitrification and nanowarming at human organ scale to enable cryopreservation

November 2024

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101 Reads

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1 Citation

Organ banking by vitrification could revolutionize transplant medicine. However, vitrification and rewarming have never been demonstrated at the human organ scale. Using modeling and experimentation, we tested the ability to vitrify and rewarm 0.5–3 L volumes of three common cryoprotective agent (CPA) solutions: M22, VS55, and 40% EG+0.6M Sucrose. We first demonstrated our ability to avoid ice formation by convectively cooling faster than the critical cooling rates of these CPAs while also maintaining adequate uniformity to avoid cracking. Vitrification success was then verified by visual, thermometry, and x-ray μCT inspection. M22 and EG+sucrose were successfully vitrified in 0.5 L bags, but only M22 was vitrified at 3 L. VS55 did not vitrify at any tested volumes. As additional proof of principle, we successfully vitrified a porcine liver (~1L) after perfusion loading with 40% EG+0.6M Sucrose. Uniform volumetric rewarming was then achieved in up to 2 L volumes (M22 with ~5 mgFe/mL iron-oxide nanoparticles) using nanowarming, reaching a rate of ~88 °C/min with a newly developed 120 kW radiofrequency (RF) coil operating at 35kA/m and 360kHz. This work demonstrates that human organ scale vitrification and rewarming is physically achievable, thereby contributing to technology that enables human organ banking.





Figure 3. Sorting SC-β cell clusters by size. (a) The SC-β cell cluster (∼220 μm in diameter), highlighted with a dashed red circle, traversed inside the sorting device. The cluster was smaller than the cutoff size (250 μm) for sorting. (b) The smaller cluster traversed in a straight line toward the channel exit. No TSAW pulse was generated to actuate the smaller cluster. Another SC-β cell cluster (∼300 μm in diameter), highlighted with a dashed green circle, also entered the device. This newly entered cluster was larger than the size cutoff for sorting. (c) The smaller cluster exited the microchannel through outlet O1. (d) A 54 ms duration TSAW pulse selectively acted upon the larger cluster. (e) The larger cluster was pushed transverse to the main flow direction. (f) The larger cluster exited the microchannel through outlet O2. Arrows on the images represent the velocity of each cluster. Inlet flow rates for inlets I1−I3 were 66, 40, and 150 μL/min, respectively. The TSAW input power was 36.5 dBm. The scale bar is 250 μm.
Figure 4. Sorting of a clump of SC-β cell clusters from a discrete SC-β cell cluster. (a) The SC-β cell cluster sample had discrete SC-β cell clusters (highlighted by a dashed red circle) as well as clumps of SC-β cell clusters (highlighted by a dashed green circle). (b) The discrete cluster (∼240 μm) was smaller than the cutoff size of 250 μm and therefore not actuated. (c) The clump of clusters was identified as a single cluster that was >250 μm in size and therefore pushed transverse to the main flow direction. The smaller discrete cluster exited the microchannel through outlet O1. (d) The clump of clusters exited the microchannel through outlet O2. The scale bar is 250 μm.
On Chip Sorting of Stem Cell-Derived β Cell Clusters Using Traveling Surface Acoustic Waves

February 2024

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38 Reads

Langmuir

There is a critical need for sorting complex materials, such as pancreatic islets of Langerhans, exocrine acinar tissues, and embryoid bodies. These materials are cell clusters, which have highly heterogeneous physical properties (such as size, shape, morphology, and deformability). Selecting such materials on the basis of specific properties can improve clinical outcomes and help advance biomedical research. In this work, we focused on sorting one such complex material, human stem cell-derived β cell clusters (SC-β cell clusters), by size. For this purpose, we developed a microfluidic device in which an image detection system was coupled to an actuation mechanism based on traveling surface acoustic waves (TSAWs). SC-β cell clusters of varying size (∼100–500 μm in diameter) were passed through the sorting device. Inside the device, the size of each cluster was estimated from their bright-field images. After size identification, larger clusters, relative to the cutoff size for separation, were selectively actuated using TSAW pulses. As a result of this selective actuation, smaller and larger clusters exited the device from different outlets. At the current sample dilutions, the experimental sorting efficiency ranged between 78% and 90% for a separation cutoff size of 250 μm, yielding sorting throughputs of up to 0.2 SC-β cell clusters/s using our proof-of-concept design. The biocompatibility of this sorting technique was also established, as no difference in SC-β cell cluster viability due to TSAW pulse usage was found. We conclude the proof-of-concept sorting work by discussing a few ways to optimize sorting of SC-β cell clusters for potentially higher sorting efficiency and throughput. This sorting technique can potentially help in achieving a better distribution of islets for clinical islet transplantation (a potential cure for type 1 diabetes). Additionally, the use of this technique for sorting islets can help in characterizing islet biophysical properties by size and selecting suitable islets for improved islet cryopreservation.




Citations (28)


... 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
Magnetic-Nanorod-Mediated Nanowarming with Uniform and Rate-Regulated Heating
  • Citing Article
  • September 2024

Nano Letters

... 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. ...

Engineering Magnetic Nanoclusters for Highly Efficient Heating in Radio-Frequency Nanowarming
  • Citing Article
  • April 2024

Nano Letters

... Researchers have effectively used different combinations of these CPAs, particularly EG and DMSO, on a variety of tissues and cells, including amnion-derived MSCs [39], cord blood [40], and embryos [41]. High concentrations of CPAs are necessary for the equilibrium vitrification process, which can potentially be detrimental to cells [42]. Consequently, the vitrification freezing method often requires both careful preparation of the CPA mixture and the gradual injection of the CPA at lower temperatures to ensure maximum safety. ...

Model-guided design and optimization of CPA perfusion protocols for whole organ cryopreservation
  • Citing Article
  • December 2023

Cryobiology

... We chose a CPA based on previous literature 11,12 and performed systematic studies to optimize the CPA loading and unloading conditions. To support rapid cooling and rewarming, we utilized a cryomesh system that we previously used to successfully cryopreserve islets 13 and other organisms 14 . Finally, we assessed PCLS viability, metabolism, function, enzymatic activity, and drug response over three days in culture. ...

Conduction‐Dominated Cryomesh for Organism Vitrification

... 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

... 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]. ...

Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model

... 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

... There are currently no volumetric means of cooling human organs, so convective cooling will be directly impacted by the size of the system, with the cooling rate at the center of the sample decreasing as the size of the system increases [20]. This may lead to insufficient cooling rates and/or physical fractures/cracking due to thermal gradients in the vitrified state [21] if a proper cooing protocol is not employed. ...

Perspective: A Guide to Successful ml to L Scale Vitrification and Rewarming

Cryoletters

... Apoptosis results when electrical pulses that are administered to cancer cells provoke thermal damage to internal structures and cell membranes [4]; it is known as inhibition of Fractal Fract. 2025, 9,34 2 of 20 proliferation on biological cells [5]. Healthy cells that are in the surrounding media are less sensitive to applied electrical pulses, avoiding significant damage. ...

Rapid joule heating improves vitrification based cryopreservation

... 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