CELL CULTURE ENGINEERING

Cell providers have streamlined their processing and isolation of conventional cell lines or even primary cells through more effective methods of procuring and handling donor tissues, improved enzymatic dissociation cocktails and techniques, and standardized cryopreservation procedures. More detailed quality control and cell health assessments have delivered consistent cell products to cell researchers

Thus, quality appears nowadays as a very important issue in all aspects of tissue culture. The quality of materials (cell lines, media and other reagents…) will greatly affect cultures quality and subsequent scientific data and products derived from them. The main areas of quality control that are of concern for tissue culture are:

  • The quality of the different reagents and materials that will be used for culture
  • Provenance of cell lines (fully characterized with DNA profile and species of origin)
  • Avoidance of cell microbial contamination (cells must be contaminant free)
  • Integrity of the cell line during culture
Cell culture

The latter point is extremely important, perhaps the most important but often a neglected one, especially where In vitro assays play a very important role such as drug discovery. Drug companies are nowadays using large testing platforms to sort out their early-stage compounds and projects, and very large amounts of time and money are riding on those decisions. The assays they use provide a simple, convenient, and rapid way to study the drugable properties of chemical entities to help advance those drug candidates. Nevertheless, all their platform results are extremely dependent on the specific properties cells display during tests running. This applies either to primary cells or to continuous cell lines that are used in that sorting.

Most of cell culture QC rely today on cell morphology without precluding any metabolic modifications that can greatly modify cell behavioral. Everybody knows that some key proteins, especially some crucial enzymes, strongly involved in cellular metabolism, are highly regulated. Some, for example, are regulated by a process called glutathionylation. Reduced glutathione (GSH) is the most prevalent biological thiol and plays a crucial role in maintaining a reduced intracellular environment. Exposure to reactive oxygen (ROS) or nitrogen species (RNS) is causatively linked to the disease pathologies associated with redox imbalance. In particular, ROS can differentially oxidize certain cysteine residues in target proteins and the reversible process of S-glutathionylation may mitigate or mediate the damage.

Mouse embryonic stem cells

Hepatotoxicity may result from exposure to any one of a number of chemical or biological stresses. Drugs, alcohol, and viruses are foremost in this regard and one common theme to the inflicted damage is the intermediate involvement of both ROS and RNS. As a consequence of its function as a detoxification organ, the liver has high cellular levels of GSH and commensurately high expression patterns of Phase I and II detoxification enzymes, particularly GSTs.

Death of hepatocytes is crucial in causing the clinical manifestations of hepatotoxicity. Changes in the redox balance after exposure to agents that deplete GSH induce apoptosis and necrosis in hepatocytes. At least one important cause/effect relationship implicates redox dysregulation of many proteins that modulate cell survival and death, these proteins being regulated either by nitrosylation or S-glutathionylation. High concentrations of damaging agents can lead to relatively large shifts in redox homeostasis resulting in necrosis of primary hepatocyte cultures. On the other hand, more modest redox alterations do not seem to promote apoptosis in hepatocytes. Any external insult, even a minor one, that will deplete GSH will also alter overall intracellular redox homeostasis and regulate the response of liver cells to TNFα or Fas-induced apoptosis. Thus, redox alterations in hepatocytes may be important in regulating the cell death pathways that mediate a broad range of different liver pathologies, independent of the causative agent. This is a very important issue when the tested drug is suspected of hepatotoxicity.

This example holds also true for cardiotoxicity or glycation toxicity in diabetes mellitus, and for all the cellular processes affected by either ROS or RNS.

This simple example clearly illustrates that, without an unambiguous definition of the oxidative stress status a cell line may have along its life, it is therefore very difficult to ascertain that cells are in the same glutathione status at day three of culture when shipment proceeds.

What concerns ROS and GSH may also concern whole cell metabolism. Cellular metabolism imbalance may have an impact on whole cell behavioral. Thus, defining a so-called “metabolic passport” to the cells that will be used as a model system to evaluate drug toxicity in safety pharmacology is strongly mandatory.

Providers using this kind of passport will be able to warn cell researchers about the intrinsic qualities of their cell lines and to propose reproducible set of cells with the same metabolic pattern. This will certainly increase the overall reliability of the results and, at the end of the process, be a true cost-effective procedure.