XenoTech receives hepatic, renal, intestinal, pulmonary, and other human tissue from various regional organ procurement organizations (OPOs) that obtain organs approved for research use. Regulations in the United States require that, regardless whether the organ is intended for transplantation or research purposes, the organ donor's identity be treated as highly confidential information. Organ procurement organizations maintain the informed consent records from each donor, and our Standard Operating Procedure requires that XenoTech personnel confirm the existence of informed consent for research purposes, prior to transport of organs to XenoTech. This procedure is intended to ensure that XenoTech manufactures human-derived products only when informed consent has been granted for research use of those specific organs. XenoTech does not, and, in consideration of confidentiality, cannot obtain the informed consent records from these organizations.

All human tissue accepted by XenoTech has been tested for the possible presence of various infectious diseases, and XenoTech does not accept human-derived material unless the donor has tested negative (non-reactive) for RPR, HIVAb, HTLVAb, HBsAg and HCVAb. All human tissue is also tested for CMVAb.  However, due to the widespread (nearly ubiquitous) appearance of CMV in the population, and its relative insignificance as an infectious agent, tissues from donors reactive for CMVAb are accepted.  The serology status of each donor is typically determined by ELISA by the organ procurement hospital.

XenoTech does not deal directly with - nor does it make any direct payments to - the surgeons who procure organs or the medical institutions where they work.

Reactions are typically carried out in 1-mL incubation mixtures that contain the following components at the final concentrations indicated in parentheses.

1. Liver microsomes (typically 0.1-1.0 mg/mL)
2. Substrate (drug or test article under investigation; various concentrations)
3. Potassium phosphate buffer (50 mM, pH 7.4)
4. Magnesium chloride (MgCl₂) (3.0 mM)
5. EDTA (1.0 mM, pH 7.4)
6. Glucose-6-phosphate (5.0 mM, pH 7.4)
7. Glucose-6-phosphate dehydrogenase (1.0 Unit/mL)
8. NADP (1.0 mM, pH 7.4)

Liver microsomes are thawed and dispensed at ~4°C. The substrate, potassium phosphate buffer, MgCl₂ and EDTA are typically combined and dispensed as a single solution at ~4°C. The substrate may need to be dissolved in organic solvent. Because organic solvents can inhibit CYP enzymes, the amount of organic solvent should be kept to a minimum (less than 10 μL/mL or 1% (v/v) of the incubation volume). If the substrate must be added in organic solvent, it should NOT be added to the microsomes directly because high concentrations of solvent can denature cytochrome P450. Substrates dissolved in inorganic solvents should be either diluted with potassium phosphate buffer/MgCl2 /EDTA solution or added after these components to avoid exposing the microsomes to high concentrations of organic solvent. These three components (also known as the NADPH-generating system), can be combined and added as a single solution. Alternatively, the three components of the NADPH-generating system can be replaced with NADPH, although this is relatively expensive.

Incubations are typically conducted at 37°C, and are stopped with a denaturant, typically organic solvent or acid. If necessary, precipitated protein is pelleted in a bench-top centrifuge, and the clear supernatant fraction is analyzed (e.g., by HPLC) for metabolites and/or remaining substrate.

It is highly recommended that the metabolism of a substrate be measured under initial rate conditions. These conditions must be determined experimentally by varying the amount of microsomal protein and incubation time to ascertain whether metabolite formation is directly proportional to time and protein concentration.

Occasionally, there is little or no information on the metabolism of the substrate with which to develop an analytical procedure. In such cases, it may be useful to incubate a fairly high concentration of pooled microsomal protein (e.g., 1mg/mL) with a high concentration of substrate (e.g., 100 µM or higher, depending on solubility) for various times (e.g., 0, 5, 10, 15, 30, 45 and 60 minutes) in order to generate sufficient quantities of metabolites for detection purposes. It should be emphasized however, that reaction phenotyping should, if at all possible, be conducted with pharmacologically relevant concentrations of substrate under initial rate conditions. It will be necessary to reevaluate the effects of protein concentration and incubation time on rates of metabolite formation if the concentration of substrate is decreased in subsequent experiments. The sum of all metabolites should constitute less than 20% of the amount of substrate present. Ideally, the amount of substrate consumed during the reaction should be less than 10% in order to measure initial rates of metabolite formation.

Once an analytical procedure has been developed and initial rate conditions have been established with the pooled sample of microsomes, the individual samples of microsomes can be examined for their ability to metabolize the compound of interest. The sample-to-sample variation in the rate of formation of each metabolite can then be compared with the activity of the individual CYP or UGT enzymes in the same microsomal samples (based on the information provided with the kit). A high correlation usually identifies the CYP or UGT enzyme responsible for generating each metabolite.