Many laboratories and companies choose to perform their own in vitro toxicological studies internally when possible, and source products such as cryopreserved hepatocytes, microsomes, subcellular fractions and plasma they use from other labs. Determining the quality of these lab products can be a tedious process.
Liver S9 subcellular fractions and cryopreserved hepatocytes are both unique products derived from the liver of organisms, though in our case, we will be specifically discussing rainbow trout as sources of these products. They each have specific criteria that determine their quality. When choosing the highest quality products for your own studies, there are many things to consider beginning with the organism itself. Below, this blog will discuss the criteria for choosing lab products and what impact they can have on your experiments.
Before a laboratory creates the products for you to use in your studies, they have to source the fish they use from hatcheries or maintain a breeding population. Though this is a part of the process that does not involve you if you don’t have fish facilities to grow them, it is no less important to the quality of the cells or S9 fractions you receive, and you should be prepared with the right questions.
Other than the recommendations from the OECD Test Guidelines 319A and 319B, there are no regulations or standards in place outlining important aspects to consider in maintenance of the fish for cell isolation or S9 fractions preparation. However, hatcheries can be certified with the presence of a veterinarian. With a veterinarian evaluating the fish, these hatcheries establish that they are a responsible source for healthy fish used for the development of cryopreserved hepatocytes or liver S9 fractions. You should ensure that the lab you purchase your products from sources their fish from certified hatcheries.
A veterinarian plays an important role guaranteeing that the fish are free of pathogens, but they are not responsible for overseeing the possible use of hormones or chemicals on fish to manipulate their growth. Some private hatcheries, for instance, will use estrogen to convert males into females (i.e. endocrine disruption), and there is no regulation against this in most countries. Male rainbow trout tend to mature earlier than females and use their energy for reproduction whereas females utilize their energy for growth. Whether fish have been manipulated with hormones or chemicals to accelerate their growth will affect their enzyme activities and the quality of the products. These fish have also grown under circumstances that don’t mimic the natural environment, altering the results of your experiments, as well.
It is the responsibility of the lab to ensure that the fish with which they develop products are healthy and chemical-free. The quality of the products you ultimately purchase from the lab relies on sourcing the right fish, and to ensure you’re choosing the right products, you should also verify the source of the fish with the lab.
The batches, or lots, of liver S9 fractions or cryopreserved hepatocytes you receive for your laboratory tests should meet a level of quality, as well. Quality, in this case, means these lots will perform as intended for your studies. The lab selling these products should properly characterize them using specific assays to ensure their quality. For S9, a proper assessment to determine protein concentration as well as basic enzyme activities must be done.
For hepatocytes, the cell concentration or density should be determined along with cell recoveries and cell viabilities. Hepatocytes are kept at very low temperatures of -150 °C with liquid nitrogen and must be thawed following a specific set of procedures. After thawing procedures, high quality hepatocytes properly handled during cryopreservation and thawing should have high recoveries, meaning you recover most of the cells in the process with high viability, ultimately recovering more healthy living cells rather than dead cells. If you have followed procedures currently, high quality cryopreserved hepatocytes should yield viabilities of >80%.
Accounting for variability within their lots, providers of these products should include cells from multiple fish for the best results, as well.
Once a lab has developed products for testing, they should characterize them to ensure their quality for customers. Pretesting their products gives you extra confidence in your experimental results. Any lab that cannot provide these results for you – or provides poor results – is not a reliable source of these products.
Phase I and Phase II Metabolic Enzyme Activity: Each lot should be evaluated for the ability to catalyze phase I and II biotransformation reactions. Each phase has a specific purpose. The purpose of characterizing for Phase I is to evaluate certain chemical reactions such as oxidation, reduction and hydrolysis. Characterizing for Phase II evaluates conjugation of the parent compound with charged groups, for example, glucuronidation and glutathione transfer to make the compound more water soluble in the liver and excreted in feces or through urine as part of the detoxification process. To evaluate metabolic enzyme activities, a lab should use proper procedures. Examples of these are Ethoxyresorufin-O-deethylase (EROD) activity, lauric acid or testosterone hydroxylation to characterize Phase I and Glutathione S-transferase (GST) of glucuronosyltransferase (UGT) for Phase II.
Pyrene: Pyrene was used in the OECD Ring Trial as a positive control or marker to indicate whether a system is acting properly. KJ Scientific not only uses pyrene as a positive control, we characterize our lots for its metabolic rates, liver intrinsic clearance and bioconcentration factor. We believe that the metabolism of a particular chemical that represents a class of chemicals, or “benchmark,” is best in some cases and a reliable indicator of activity when EROD basal levels are quite low. There may be a lot of variability between labs when testing using such assays.
As we advance the science, more and more tests will be standardized and used for the characterization of subcellular fractions and cells.
Over time, the accumulation of the historical data in the lab acquired through testing can be used to establish an acceptance criteria, the set of conditions that each lot must satisfy to ensure that it is functioning as expected.
Liver S9 fractions and cryopreserved hepatocytes must have enzyme activities that fall within the acceptance criteria. Acceptance criteria is usually supplied by the lab producing these products and is based on their historical data. However, you can also compare it to data from other sources (published but reliable data). It is up to you as the consumer to ensure the company you purchase your lab products from does a proper characterization of them or you must take it upon yourself to do so. Without this data, you will not know if the products are of poor quality, and poor quality S9 fractions or cells will make the results of any experiments you perform with them less accurate.
At KJ Scientific, for instance, we use the metabolic rates of pyrene as a marker or part of the characterization of the product. Over time, we have accumulated significant data on its metabolic rates, and this cumulative historical data can be used as an indicator for us. When we do an experiment with pyrene as the positive control and compare the results of the metabolic rate to our historical data, we can determine if it meets our acceptance criteria if it falls within the range of results we had in the past. From this, we can say with confidence that the products are performing as expected.
You should also ensure that the products are prepared by knowledgeable experts using proper methodologies. The processes required in preparing liver S9 fractions and cryopreserved hepatocytes call for the use of the latest technology and responsible lab practices to do them accurately. If the lab does not know what they’re doing regarding the use of proper technology and proper procedures, the resulting products – and your experiments – will not be as reliable.
The process of cell isolation is a very labor intensive procedure. In order to do it following proper guidelines, the fish have to be euthanized properly to avoid suffering or harm to the animal as much as possible and following animal care guidelines (e.g. those of the (IACUC) Institutional Animal Care and Use Committee). This procedure can only be performed by qualified personnel, and they need the proper facilities. The process of isolating the liver cells must also be done carefully and precisely. If it is the practice of the lab to isolate many cells from too many fish at once, the resulting hepatocytes will not be as high quality. Proper technologies should also be employed. To make S9 fractions, for example, liver cells must be properly and carefully perfused, homogenized and undergo centrifugation. Make sure that the lab products you purchase are using correct procedures and technologies.
Proper cryopreservation of hepatocytes also requires expertise to perform correctly. It is a lengthy process that involves breaking the cell junction to separate cells while their membranes remain intact. Then the cells must be washed following specific procedures before ingredients can be added that contain reagents to prevent icicles from forming during freezing. The formation of icicles can cause cell membranes to rupture. S9 fractions are transported in dry ice and cryopreserved hepatocytes in cryoshippers with liquid nitrogen vapors for shipment to customers anywhere in the world.
The lab should be able to instruct you on procedures for correctly thawing the products when you need them. After all, they should want you to know how to properly handle their products or it could affect their quality and the confidence you have in them.
The labs at KJ Scientific have the experience and expertise in this field to deliver the best lab test products on the market. Coming from the pharmaceutical industry and having worked extensively with human liver cells, we know what we’re doing regarding proper procedures and best practices. We use the most advanced technology and techniques that follow the most current guidelines to ensure we produce our products safely and accurately. Our lots have higher recoveries compared to other sources and high viabilities, ensuring that you will have the resources you need for your experiments when you need them, and they will function appropriately.
KJ Scientific can ship our products right to you, or better yet, we can conduct your experiments for you at our labs and deliver you the results. If you’d like to learn how KJ Scientific can work with you on your toxicological in vitro metabolism studies, request a webinar today!