Name: Video: Using a Chemical Biopsy for Graft Quality Assessment
Uploaded: 2020-06-17T13:30:00.000+00:00
Duration: 5 min

The study was supported by grant Opus UMO-2017/27/B/NZ5/01013 from National Science Centre. The authors would like to acknowledge MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany for providing SPME devices. The life science business of Merck operates as MilliporeSigma in the U.S. and Canada. Also, the authors want to thank Thermo Fisher Scientific for the access to Q-Exactive Focus orbitrap mass spectrometer. The authors would like to thank Dr. Aleksandra Woderska-Jasin&#769;ska and the personnel of the Department of Transplantology and General Surgery in Bydgoszcz for their kind assistance in the project. BB want to thank Prof. Janusz Pawliszyn for the opportunity of sample collection at Toronto General Hospital during her stay at University of Waterloo.

All animals received humane care in compliance with the &#8216;&#8216;Principles of Laboratory Animal Care&#8217;&#8217; formulated by the National Society for Medical Research and the &#8216;&#8216;Guide for the Care of Laboratory Animals&#8217;&#8217; published by the National Institute of Health, Ontario, Canada. The Animal Care Committee of the Toronto General Research Institute approved all studies. The research involved human subjects was approved by the Bioethical Committee at Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Torun.
NOTE: Obtain approval from appropriate ethical boards. Remember to always wear safety gloves. Do not touch the extraction phase of SPME probes. The use of deactivated glass vials is recommended for lipidomic analyses.
1. Preparation of probes
<ol>
	<li>Prepare titanium-nickel alloy probes (40 mm length; 0.2 mm diameter) coated with 7 mm mix-mode sorbent. The number of probes depends on the time points targeted during the entire procedure and the number of replicates (3 is recommended per time point). 
	NOTE: The length and type of extraction phase may be adjusted based on the mode of study, the polarity of metabolites, and the sample matrix.</li>
	<li>Prepare a cleaning mixture composed of 2:1 chloroform:methanol (v/v). Pipet 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial. 
	NOTE: Before use, clean all probes to remove contaminating particles.</li>
	<li>Put the vials on the agitator and set the agitation speed at 1,200 rpm. After 45 min, stop the device and rinse the coatings with LC-MS grade water.</li>
	<li>As coatings must undergo a preconditioning step to activate them, prepare a preconditioning mixture composed of 1:1 methanol:water (v/v). Pipet 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial.</li>
	<li>Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm.</li>
	<li>After 60 min, stop the agitator and rinse the coatings with LC-MS grade water.</li>
	<li>Sterilize probes according to the standard surgical equipment sterilization protocol.</li>
</ol>
2. Extraction
<ol>
	<li>Open the sterile package right before sampling to ensure a sterile environment.</li>
	<li>Insert two probes directly into the kidney cortex for 10 min at each time point. The entire length of the coating must be covered by the tissue matrix; no specific angle is required, but ca. 90 deg is usually used. 
	NOTE: The entire medical procedure follows standard protocols in given institution. No modification concerning SPME sampling is considered. The procedure involves the six following sampling time points: 
	a) before kidney resection, in vivo from donor; 
	b)-e) after 1 h, 3 h, 5 h, 7 h of kidney perfusion, ex vivo in organ chamber; 
	f) after reperfusion, in vivo from recipient.</li>
	<li>Retract the probes by pulling it out from the tissue and then rinse coatings with LC-MS grade water to clear any remaining blood from the coating surface. Rinse away from the surgical site, and immediately after removal of the probes.</li>
</ol>
3. Transport and storage
<ol>
	<li>Place probes in separate vials and close them.</li>
	<li>Place vials in a Styrofoam box filled with dry ice or in liquid nitrogen for transport.</li>
	<li>Store samples in a freezer (-80 &#176;C), or immediately commence the desorption step.</li>
</ol>
4. Desorption
<ol>
	<li>Prepare desorption solutions composed of 80:20 acetonitrile:water (v/v) for metabolomic analysis, and 1:1 isopropanol:methanol (v/v) for lipidomic analysis.</li>
	<li>Pipet 100 &#956;L of the solution to inserts placed into 2.0 mL labeled vials and place one probe, previously pierced through the cap, in each vial.</li>
	<li>Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm for 120 min.</li>
	<li>Remove probes from the vials. Obtained extracts are now ready for instrumental analysis.</li>
</ol>
5. LC-MS analysis
<ol>
	<li>Place vials containing extracts in the autosampler of the LC-MS system. 
	NOTE: Liquid chromatography (RP, HILIC) coupled with high resolution mass spectrometry and an orbitrap mass analyzer were used for this study. For metabolomic analysis parameters, go to step 5.2. For lipidomic analysis parameters, go to step 5.6.</li>
	<li>Use a pentafluorophenyl (PFP) column (2.1 mm x 100 mm, 3 &#956;m) for reversed phase separation.
	<ol>
		<li>Set the flow rate to 300 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 25 &#176;C, respectively.</li>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: water:formic acid (99.9:0.1, v/v), and mobile phase B: acetonitrile:formic acid (99.9:0.1, v/v). Set the mobile phase flow according to the following parameters: starting mobile phase flow (0-3 min): 100% A followed by a linear gradient to 10% A (3-25 min), ending with isocratic flow of 10% A until 34 min, followed by 6 min of column re-equilibrium time.</li>
	</ol>
	</li>
	<li>For HILIC separation, use a HILIC column (2.0 mm x 100 mm, 3 &#956;m, 200A). Set the flow rate to 400 &#956;L/min.
	<ol>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: acetonitrile:ammonium acetate buffer (9:1, v/v, effective salt concentration 20 mM), mobile phase B: acetonitrile:ammonium acetate buffer (1:1, v/v, effective salt concentration 20 mM).</li>
		<li>Set the mobile phase flow according to the following parameters: starting mobile phase flow (0-3 min) at 100% A, hold for 3.0 min and then ramp to 100% B within 5 min. Hold with 100% B until 12 min, followed by 8 min of column re-equilibrium time.</li>
	</ol>
	</li>
	<li>Set the scan range to 85-1000 m/z. Set HESI ion source parameters in positive ionization mode to: spray voltage 1,500 V, capillary temperature 300 &#176;C, sheath gas 40 a.u., aux gas flow rate 15 a.u., probe heater temperature 300 &#176;C, S-Lens RF level 55%.</li>
	<li>Run QC samples composed of 10 &#181;L of each analyzed sample (regularly, every 10-12 samples) to monitor instrument performance.</li>
	<li>For reversed phase separation, use a C18 column (2.1 mm x 75 mm, 3.5 &#956;m).
	<ol>
		<li>Set the flow rate to 200 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 55 &#176;C, respectively. Prepare mobile phases according to the following proportions: mobile phase A: H2O:MeOH (60:40, v/v), 10 mM ammonium acetate and 1 mM acetic acid; mobile phase B: IPA:MeOH (90:10, v/v), 10 mM ammonium acetate and 1 mM acetic acid.</li>
		<li>Set the mobile phase flow according to the following parameters: 0-1 min (20% B), 1-1.5 min (20-50% B), 1.5-7.5 min (50-70% B), 7.5-13 min (70-95% B), 13-17 min (95% B), 17-17.1 min (95-20% B), 17.1-23 min (20%).</li>
	</ol>
	</li>
	<li>For HILIC separation, use a HILIC column (100 x 2.1 mm, 3 &#956;m).
	<ol>
		<li>Set the flow rate to 400 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 40 &#176;C, respectively.</li>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: ACN; mobile phase B: 5 mM ammonium acetate in water. Set the mobile phase flow according to the following parameters: 0-2 min (96% B), 2-15 min (96-80% B), 15-15.1 min (80-96% B), 15.1-21 min (96% B).</li>
	</ol>
	</li>
	<li>Set HESI ion source parameters in positive ionization mode to spray voltage 3,500 V, capillary temperature 275 &#176;C, sheath gas 20 a.u., aux gas flow rate 10 a.u., probe heater temperature 300 &#176;C, S-Lens RF level 55%.</li>
	<li>Run QC samples composed of 10 &#181;L of each analyzed sample (every 10-12 samples) to monitor instrument performance.</li>
	<li>Perform data acquisition in software compatible with the instrument.</li>
</ol>
6. Data analysis
<ol>
	<li>Perform data processing, putative identification, and statistical analysis with the use of software dedicated to untargeted metabolomics and lipidomics analysis. 
	NOTE: Principal components analysis (PCA) and box-whisker plots can be obtained to visualize data structure.</li>
</ol>

<ol><li>U.S. Department of Health & Human Services. Organ Procurement and Transplantation Network. , Available from: 
 <a target="_blank" href="https://optn.transplant.hrsa.gov">https://optn.transplant.hrsa.gov</a> (2019).</li>
<li>Branger, P., Undine, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBranger%2C+P+%22Annual+Report+2018%2FEurotransplant+International+Foundation%22">Annual Report 2018/Eurotransplant International Foundation</a>. , Eurotransplant Foundation. Leiden. (2018).</li>
<li>Dare, A. J., Pettigrew, G. J., Saeb-Parsy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preoperative+assessment+of+the+deceased-donor+kidney%3A+From+macroscopic+appearance+to+molecular+biomarkers%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Preoperative assessment of the deceased-donor kidney: From macroscopic appearance to molecular biomarkers.</a> Transplantation. 97, 797-807 (2014).</li>
<li>Mueller, T. F., Solez, K., Mas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+kidney+organ+quality+and+prediction+of+outcome+at+time+of+transplantation%5BTitle%5D)+AND+%22Seminars+in+Immunopathology%22%5BJournal%5D)">Assessment of kidney organ quality and prediction of outcome at time of transplantation.</a> Seminars in Immunopathology. 33, 185-199 (2011).</li>
<li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipidomics+comparing+DCD+and+DBD+liver+allografts+uncovers+lysophospholipids+elevated+in+recipients+undergoing+early+allograft+dysfunction%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Lipidomics comparing DCD and DBD liver allografts uncovers lysophospholipids elevated in recipients undergoing early allograft dysfunction.</a> Scientific Reports. 5, 1-10 (2015).</li>
<li>Rao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+lipid+changes+in+acute+kidney+injury+using+SWATH+lipidomics+coupled+with+MALDI+tissue+imaging%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Renal+Physiology%22%5BJournal%5D)">Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.</a> American Journal of Physiology - Renal Physiology. 310, 1136-1147 (2016).</li>
<li>Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolomics%3A+The+principles+and+potential+applications+to+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">Metabolomics: The principles and potential applications to transplantation.</a> American Journal of Transplantation. 5, 2814-2820 (2005).</li>
<li>Kim, S. J., Kim, S. H., Kim, J. H., Hwang, S., Yoo, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+metabolomics+in+biomedical+research%5BTitle%5D)+AND+%22Endocrinology+and+Metabolism%22%5BJournal%5D)">Understanding metabolomics in biomedical research.</a> Endocrinology and Metabolism. 31, 7-16 (2016).</li>
<li>Bojko, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solid+phase+microextraction+fills+the+gap+in+tissue+sampling+protocols%5BTitle%5D)+AND+%22Analytica+Chimica+Acta%22%5BJournal%5D)">Solid phase microextraction fills the gap in tissue sampling protocols.</a> Analytica Chimica Acta. 803, 75-81 (2013).</li>
<li>Filipiak, W., Bojko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SPME+in+clinical%2C+pharmaceutical%2C+and+biotechnological+research+-+How+far+are+we+from+daily+practice%5BTitle%5D)+AND+%22TrAC+-+Trends+in+Analytical+Chemistry%22%5BJournal%5D)">SPME in clinical, pharmaceutical, and biotechnological research - How far are we from daily practice.</a> TrAC - Trends in Analytical Chemistry. , 115-213 (2019).</li>
<li>Bojko, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+invasive+in+vivo+tissue+sampling+for+monitoring+biomarkers+and+drugs+during+surgery%5BTitle%5D)+AND+%22Laboratory+Investigation%22%5BJournal%5D)">Low invasive in vivo tissue sampling for monitoring biomarkers and drugs during surgery.</a> Laboratory Investigation. 94, 586-594 (2014).</li>
<li>Ahmad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biopsy+of+the+transplanted+kidney%5BTitle%5D)+AND+%22Seminars+in+Interventional+Radiology%22%5BJournal%5D)">Biopsy of the transplanted kidney.</a> Seminars in Interventional Radiology. 21, 275-281 (2004).</li>
<li>Plattner, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complications+and+adequacy+of+transplant+kidney+biopsies%3A+A+comparison+of+techniques%5BTitle%5D)+AND+%22Journal+of+Vascular+Access%22%5BJournal%5D)">Complications and adequacy of transplant kidney biopsies: A comparison of techniques.</a> Journal of Vascular Access. 19, 291-296 (2018).</li>
<li>Tapia-Canelas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complications+associated+with+renal+graft+biopsy+in+transplant+patients%5BTitle%5D)+AND+%22Nefrologia%22%5BJournal%5D)">Complications associated with renal graft biopsy in transplant patients.</a> Nefrologia. 34, 115-119 (2014).</li>
<li>Afshinnia, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipidomics+and+Biomarker+Discovery+in+Kidney+Disease%5BTitle%5D)+AND+%22Seminars+in+Nephrology%22%5BJournal%5D)">Lipidomics and Biomarker Discovery in Kidney Disease.</a> Seminars in Nephrology. 38, 127-141 (2018).</li>
<li>Zhao, Y. Y., Vaziri, N. D., Lin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipidomics%3A+New+insight+into+kidney+disease%5BTitle%5D)+AND+%22Advances+in+Clinical+Chemistry%22%5BJournal%5D)">Lipidomics: New insight into kidney disease.</a> Advances in Clinical Chemistry. 68, 153-175 (2015).</li>
<li>Kaminski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+Consumption+by+Warm+Ischemia-Injured+Porcine+Kidneys+in+Hypothermic+Static+and+Machine+Preservation%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Oxygen Consumption by Warm Ischemia-Injured Porcine Kidneys in Hypothermic Static and Machine Preservation.</a> Journal of Surgical Research. 242, 78-86 (2019).</li>
<li>Wijermars, L. G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+postreperfusion+metabolic+recovery+directly+associates+with+incident+delayed+graft+function%5BTitle%5D)+AND+%22Kidney+International%22%5BJournal%5D)">Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.</a> Kidney International. 90, 181-191 (2016).</li>
<li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteo-metabolomics+reveals+compensation+between+ischemic+and+non-injured+contralateral+kidneys+after+reperfusion%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Proteo-metabolomics reveals compensation between ischemic and non-injured contralateral kidneys after reperfusion.</a> Scientific Reports. 8, 1-12 (2018).</li>
<li>Solati, Z., Edel, A. L., Shang, Y., Karmin, O., Ravandi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidized+phosphatidylcholines+are+produced+in+renal+ischemia+reperfusion+injury%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Oxidized phosphatidylcholines are produced in renal ischemia reperfusion injury.</a> PLoS One. 13, 1-24 (2018).</li>
<li>Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolomics+in+monitoring+kidney+transplants%5BTitle%5D)+AND+%22Current+Opinion+in+Nephrology+and+Hypertension%22%5BJournal%5D)">Metabolomics in monitoring kidney transplants.</a> Current Opinion in Nephrology and Hypertension. 15, 637-642 (2006).</li>
<li>Abbiss, H., Maker, G. L., Trengove, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolomics+approaches+for+the+diagnosis+and+understanding+of+kidney+diseases%5BTitle%5D)+AND+%22Metabolites%22%5BJournal%5D)">Metabolomics approaches for the diagnosis and understanding of kidney diseases.</a> Metabolites. 9, (2019).</li>
<li>Zhang, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolomics+insights+into+chronic+kidney+disease+and+modulatory+effect+of+rhubarb+against+tubulointerstitial+fibrosis%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Metabolomics insights into chronic kidney disease and modulatory effect of rhubarb against tubulointerstitial fibrosis.</a> Scientific Reports. 5, 1-17 (2015).</li>
<li>Looby, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solid+phase+microextraction+coupled+to+mass+spectrometry%3A+Via+a+microfluidic+open+interface+for+rapid+therapeutic+drug+monitoring%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Solid phase microextraction coupled to mass spectrometry: Via a microfluidic open interface for rapid therapeutic drug monitoring.</a> Analyst. 144, 3721-3728 (2019).</li>
<li>Gómez-Ríos, G. A., Tascon, M., Pawliszyn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coated+blade+spray%3A+Shifting+the+paradigm+of+direct+sample+introduction+to+MS%5BTitle%5D)+AND+%22Bioanalysis%22%5BJournal%5D)">Coated blade spray: Shifting the paradigm of direct sample introduction to MS.</a> Bioanalysis. 10, 257-271 (2018).</li>
</ol>

The authors would like to acknowledge MilliporeSigma (Merck KGaA, Darmstadt, Germany) for providing SPME devices and Thermo Fisher Scientific for access to the Q-Exactive Focus Orbitrap mass spectrometer.

Evaluation of organ quality remains a large challenge for physicians, who must make quick informed decisions regarding whether a given organ is viable for transplantation or whether it must be discarded. Multiple factors, such as donor age, duration of ischemia, and infections and inflammatory processes, can affect the long-term graft outcome. While diverse methods have been developed to date to diagnose kidney allograft function, histopathological inspection remains the gold standard in that matter3,4,12. Although the biopsy procedure can yield significant information regarding pre-existing donor disease and vascular changes, it is not free of flaws. Sampling errors associated to interobserver variability and sampling of insufficient glomeruli for comprehensive information regarding organ function remain typical concerns in this regard. Moreover, specimen preparation brings some issues such as an incomplete appraisal of the graft in case of frozen sections, and extension of procedure time for paraffin sectioning. However, the increased risk of hemorrhage, which may appear acutely as microscopic or gross hematuria, is the major life-threatening complication associated with the biopsy procedure. For this reason, the number of allowable biopsies is strictly limited in transplantation procedures, a factor that hampers the capture of dynamic changes and time series analyses via this method12,13,14. The benefits of a histological analysis must be weighed against the risks associated with the methodology. The value of histological findings is indisputable, but they do not explain the molecular mechanisms of the aberrations.
Metabolomics and lipidomics are the youngest domains of the &#8220;-omics&#8221; scientific family. The complete set of low molecular (&#60;1,200 Da) human metabolites and lipids connected within a metabolic network is defined as a human metabolome. The genome remains relatively constant throughout its lifetime, with slight modifications caused by mutations occurring infrequently. The metabolome is the product of gene expression, which is highly sensitive to changes in all biological processes as well as environmental factors. The dynamic nature of metabolites and lipids makes them perfect indicators of current organ condition7,8,15,16. The SPME method proposed in the abovementioned protocol enables detection of changes occurring in the organ during its preservation, starting from organ removal from the donor&#8217;s body till revascularization at the recipient&#8217;s. The small diameter of the probe (~200 &#181;m) provides minimal invasiveness and allows for several samplings from the same organ without causing any damage to the tissue. Conducting studies using kidney, as the most frequently transplanted organ, allows for a better understanding and further characterization of the metabolic pathways responsible for declining the quality and function of grafts. The possibility of monitoring modifications over time certainly is an important advantage of the technique as compared to conventional invasive methods such as biopsy. The currently presented analysis identified altered concentrations of various groups of lipids and metabolites, especially of essential amino acids, purines, purine nucleosides, and glycerophospholipids. These results are consistent with previous tissue analysis reports5,6,17,18,19,20. To date, the majority of scientific reports utilizing metabolomics or lipidomics to explain processes inducing complications after transplantation or ischemia/reperfusion injury (IRI) phenomena have been limited to analysis of biofluids21,22,23.
Each clinical application requires optimization of the sampling protocol to ensure that the performance of the analytical method meets expected criteria. In this regard, the benefit of utilizing SPME is the possibility of adjusting conditions for various experimental designs. The variety of accessible extraction phases provides a broad spectrum of extracted metabolites with diversified polarities. At the same time, this might be considered as a limitation of the method due to the fact that each sorbent provides selectivity towards specific features and does not extract all compounds present in the sample matrix. It should be noted that SPME coatings extract only via free molecules, and simply do not interact with a bound fraction of the analyte. The biocompatibility of the coatings does not introduce toxicity to the tissue while restraining the extraction of large molecules such as proteins; as a consequence, the enzymatic processes are inhibited already at the stage of sample collection and the presence of artefacts is minimized, which is a great advantage over alternative sampling methods. The length of the coating influences the efficiency of extraction (i.e., the length of the coating designates the surface area and the extraction phase volume); thus, longer coatings yield higher recoveries. On the other hand, shorter coatings enable higher spatial resolution. For reliable results, it is crucial to submerge the probe to the exact same depth of kidney cortex. Insertion too deep causes the risk of entering the kidney medulla. The time of the extraction is also proportional to the extraction efficiency. Therefore, selection of optimum extraction time is one of the most critical steps in SPME method development. The accuracy of time measurement provides the highest repeatability. In biological applications such as the one discussed, there is always a compromise between the sensitivity and repeatability of the analytical protocol and the restrictions of the medical procedure. While equilibrium extraction provides the highest sensitivity, for safety reasons, pre-equilibrium conditions are often used in such applications, as extraction time should not affect the total duration of the surgery. The efficiency of desorption is determined by the time of the process and the composition of the desorption solvent, which should be compatible with the mobile phase used for chromatographic separation9,10,11.
One of the major requirements for diagnostic instrumentation used for intra-surgical assessments is time of analysis. Current attempts are being made to develop a rapid tool for in vivo SPME extraction coupled directly to a mass spectrometer via microfluidic open interface (MOI)24 or coated blade spray (CBS)25. Such approaches would allow for the disclosure of analytical results in real or close to real time. The use of such methods for pre-intervention analyses of metabolic and lipidomic profiles could enhance the decision-making process during transplantation procedures, enabling the best possible personalized approach and fast response in case of organ failure.
As a summary, it is hypothesized that the proposed protocol will enable attainment of full metabolic and lipidomic profiles of kidney grafts, which in turn would provide a comprehensive assessment of organ quality and characterization of the processes responsible for ischemia-reperfusion injury. The novelty of the project includes the utilization of solid-phase microextraction (SPME), offering low invasive sampling of living systems, in combination with one of the most innovative technologies available for metabolomics and lipidomics analysis (e.g., the Orbitrap high resolution mass spectrometer). SPME combines sample collection, extraction, and quenching of metabolites in one step, therefore making it a perfect tool for rapid analysis. It is expected that this protocol will help to answer questions related to what pre-transplant conditions of the kidney are responsible for delayed organ function or its dysfunctions after transplantation, as well as to how the graft preservation protocol influences the biochemistry of the organ. Such knowledge would not only have significant impact on the prevention of possible complications related to transplantation, but may help to improve current graft preservation protocols, minimizing loss of viable transplant tissue as well as loss of life. The proposed solution will open the door to further investigations in this field, including the validation of specific potential biomarkers and improvement of therapeutic outcomes in transplantology.

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60946/using-a-chemical-biopsy-for-graft-quality-assessment">Using a Chemical Biopsy for Graft Quality Assessment</a>. One of the affiliations was&#160;updated.
The third affiliation was&#160;updated from:
Department of Transplantation and General Surgery, University Hospital, University of Nicolaus Copernicus Torun&#34;
to:
Department of Transplantology and General Surgery, Collegium Medicum in Bydgoszcz, Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60946/using-a-chemical-biopsy-for-graft-quality-assessment">Using a Chemical Biopsy for Graft Quality Assessment</a>. One of the affiliations was&#160;updated.

Erratum: Using a Chemical Biopsy for Graft Quality Assessment

According to the United States&#8217; Organ Procurement and Transplantation Network, there were 94,756 patients waiting for kidney transplants in the US in 2019; while in Europe in 2018, that number was 10,791. Every ten minutes, someone is added to the national transplant waiting list in the U.S., and it is estimated that 20 people die each day waiting for a transplant1,2. Kidney transplantation is a life-saving treatment for a large number of people suffering with end-stage renal dysfunction worldwide. The procedure is associated with increased survival rate and greater quality of life when compared to conventional dialysis.
However, transplantation faces many serious problems, such as organ shortage or lack of effective tools for organ quality assessment. The standard protocols are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information regarding the quality of the graft. While a visual appraisal allows for identification of tumors visible to the eye, anatomical abnormalities, or extensive damage to the grafts, this approach is very subjective, varying in its effectiveness according to the experience of the observers. Biopsy, on the other hand, can provide valuable information regarding pre-existing renal disorders, and is thus considered to be a method of objective and evidenced value in determining graft outcomes. However, the biopsy procedure is not free of flaws; there is the risk of potential complications such as bleeding and an additional 4-5 hours of sample preparation is required, which significantly prolongs the cold ischemic time. Therefore, especially in Europe, the use of direct tissue analysis is limited to expanded criteria donors (ECD) and donors after circulatory death (DCD)3,4.
Metabolomics and lipidomics have been recently recognized as promising approaches to attaining a better understanding of the changes in biochemical pathways occurring during organ preservation. Metabolomic and lipidomic profiling enables monitoring of immediate responses of the system to sudden environmental changes related to organ removal with subsequent consequences: ischemia, oxidative stress, or inflammatory responses5,6,7,8. The kidney is an organ that is largely associated with metabolic processes, thus measurements of metabolites and lipids concentrations may permit identification of potential organ quality biomarkers and enable better predictions of graft outcome.
Given the above complications and limitations associated with current organ quality assessment methods, a less invasive diagnostic solution is needed for quick and complex organ quality assessment. Solid phase microextraction (SPME) complies with these requirements as a minimally invasive analytical method that enables coverage of a broad spectrum of metabolites and lipids. The technique is based on the insertion of a thin (~200 &#181;m), biocompatible, titanium-nickel alloy probe covered with a selective extraction phase into the examined organ for a short time. It should be emphasized that SPME prevents protein extraction, and hence enables metabolism inhibition already at the stage of sample collection, which is a significant advantage over alternative methods. Moreover, the miniaturization of the device allows for the execution of repetitive and simultaneous analyses of few structures of the organ9,10,11.

<table><tbody><tr><td>Acetic acid</td><td>Merck</td><td>5330010050</td><td>Mobile phase additive</td></tr><tr><td>Acetonitrile</td><td>Alchem</td><td>696-34967-4X2.5L</td><td>HPLC solvent</td></tr><tr><td>Ammonium acetate</td><td>Merck</td><td>5330040050</td><td>Mobile phase additive</td></tr><tr><td>BENCHMIXER XL MULTI-TUBE VORTEXER</td><td>Benchmark Scientific</td><td>BV1010</td><td>Vortex mixer</td></tr><tr><td>Caps</td><td>Perlan Technologies</td><td>5183-2076</td><td>Blue scrw tp, pre-slit PTFE/Si spta, 100PK</td></tr><tr><td>Chloroform</td><td>Merck</td><td>1024441000</td><td></td></tr><tr><td>Discovery HS F5 Supelguard Cartridge, 3 &amp;mu;m, L &amp;times; I.D. 2 cm &amp;times; 2.1 mm</td><td>Merck</td><td>567570-U</td><td>HPLC guard column</td></tr><tr><td>Discovery HS F5, 2.1 mm x 100 mm, 3 &amp;mu;m</td><td>Merck</td><td>567502-U</td><td>HPLC column</td></tr><tr><td>Formic acid</td><td>Alchem</td><td>497-94318-50ML</td><td>Mobile phase additive</td></tr><tr><td>Glass vials</td><td>Perlan Technologies</td><td>5182-0714</td><td></td></tr><tr><td>HILIC Luna 3 &amp;mu;m, 200A, 100 x 2.0 mm</td><td>Shim-Pol</td><td>PHX-00D-4449-B0</td><td>HPLC column</td></tr><tr><td>HILIC SecurityGuard Cartridge, 3 &amp;mu;m, 4 x 2.0 mm</td><td>Shim-Pol</td><td>PHX-AJ0-8328</td><td>HPLC guard column</td></tr><tr><td>Isopropanol</td><td>Alchem</td><td>231-AL03262500</td><td>HPLC solvent</td></tr><tr><td>Methanol</td><td>Alchem</td><td>696-34966-4X2.5L</td><td>HPLC solvent</td></tr><tr><td>Nano-pure water</td><td>Merck</td><td>1037281002</td><td>HPLC solvent</td></tr><tr><td>Q Exactive Focus hybrid quadrupole-Orbitrap MS</td><td>Thermo Scientific</td><td>Q Exactive Focus</td><td>Mass Spectrometer</td></tr><tr><td>SeQuant ZIC-cHILIC 3&amp;micro;m,100&amp;Aring; 100 x 2.1 mm</td><td>Merck</td><td>1506570001</td><td>HPLC column</td></tr><tr><td>SeQuant ZIC-HILIC Guard Kit 20 x 2.1 mm</td><td>Merck</td><td>1504360001</td><td>HPLC guard column</td></tr><tr><td>SPME LC fiber probes, mixed mode</td><td>Supelco</td><td>prototype fibers</td><td></td></tr><tr><td>UltiMate 3000 HPLC systems</td><td>Thermo Scientific</td><td>UltiMate 3000</td><td>HPLC system</td></tr><tr><td>Vial inserts (deactivated)</td><td>Perlan Technologies</td><td>5181-8872</td><td></td></tr><tr><td>XSelect CSH C18 3.5&amp;mu;m 2.1x75mm</td><td>Waters</td><td>186005644</td><td>HPLC column</td></tr><tr><td>XSelect CSH C18 VanGuard Cartridge 3.5&amp;mu;m, 2.1x5mm</td><td>Waters</td><td>186007811</td><td>HPLC guard column</td></tr></tbody></table>

using a chemical biopsy for graft quality assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

Sample collection was performed according to the SPME method described above, using probes coated with a 7 mm mixed-mode extraction phase. Sampling was conducted directly from the graft tissue (in vivo before transplantation), ex vivo in kidney chamber after 1 h, 3 h, 5 h, and 7 h of perfusion, and in vivo after revascularization in the recipient. Untargeted metabolomic and lipidomic analyses were carried out with the use of liquid chromatography (RP, HILIC) coupled with high resolution mass spectrometry, with the mass analyzer set in positive ionization mode. To assess data quality and attain general insights regarding the results, data was subjected to principal component analysis (PCA). As shown in Figure 1, QC samples formed a tight cluster, confirming the quality of the analyses. The studied groups exhibit relatively good separation, allowing for visualization of differences in metabolic and lipidomic profiles before and after transplantation, as well as during organ perfusion (Figure 2). SPME sampling permits metabolic profiling of the organ over time. Box-whisker plots of selected metabolites serve to exemplify alterations in metabolite levels throughout the experiment (Figure 3). The wide spectrum of extracted features separated on both RP and HILIC columns is shown in Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60946/60946fig01.jpg" /> 
Figure 1: Principal component analysis showing clustering of quality control samples for metabolomic reversed-phase (A), HILIC (B) and lipidomic reversed-phase (C), HILIC (D) analyses.&#160;The quality control is necessary in untargeted analysis to monitor any possible changes induced by instrumental instability. Tight cluster of QC samples ensures that all changes observed are of biological origin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60946/60946fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60946/60946fig02.jpg" /> 
Figure 2: Principal component analysis showing metabolomic (A) and lipidomic (B) differences between particular sampling points.&#160;Untargeted profiling of kidney with SPME-LC-HRMS enables to observe biochemical differences in organs at subsequent steps of their preservation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60946/60946fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60946/60946fig03.jpg" /> 
Figure 3: Box-whisker plots of selected compounds showing alterations in metabolites (A, B) and lipids (C, D) during the peritransplant period.&#160;After selection and identification of discriminating compounds box-whisker plots enables to monitor changes in levels of these compounds at subsequent steps of the protocol and compare the metabolites levels in organs subjected to different preservation protocols or harvested from different donors e.g. heart beating donors or donors after cardiac death. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60946/60946fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60946/60946fig04.jpg" /> 
Figure 4: Ion map (m/z versus retention time) of features obtained by LC-MS analysis with (A) reversed-phase and (B) HILIC separation.&#160;The plot enables to estimate overall number of features obtained with the proposed protocol (from extraction to detection) and assess analyte coverage based on the polarity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60946/60946fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using a Chemical Biopsy for Graft Quality Assessment at JoVE.com

Using a Chemical Biopsy for Graft Quality Assessment

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

using-a-chemical-biopsy-for-graft-quality-assessment

Nanyang Technological University

Research

JoVE Journal

Medicine

5.1K Views.  Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz. Chemische biopsie is een nieuwe diagnostische oplossing in orgaantransplantatie voor een snelle en complexe graft kwaliteitbeoordeling. Momenteel is er geen andere lage invasieve methoden beschikbaar die een dergelijke analyse mogelijk maken. Het belangrijkste voordeel van deze techniek is de mogelijkheid van het monitoren van graft weefsel wijzigingen na verloop van tijd van behoud als gevolg van de eenvoud en lage invasieve vermogen van SPME sondes.De kleine diameter van de sonde en ...

Video: Using a Chemical Biopsy for Graft Quality Assessment

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Immunology and Infection

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Cancer Research

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Bioengineering

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Using a Chemical Biopsy for Graft Quality Assessment

Samenvatting

Meer video's verkennen