A bioassay is essentially an analytical process by which the potency or concentration of a substance is determined, by means of the effect that it has on living cells or tissues. A bioassay can be used to get a reasonably accurate estimate of the potency of an agent, by observing, over a period of time, its effects on living animal and human tissues. In general, bioassays are utilized to make a quality assessment of various products or to detect any imminent biological hazards. They may also be used to determine the potential environmental impact of new technology or facility.
Sometimes, bioassays – also known as cell-based assays – do not perform as well as expected. A reputed laboratory with years of experience in performing cell-based assays should ideally be chosen for the task, as over the years they have developed an awareness of the processes that can improve the performance of a bioassay.
Five of the factors that can improve the performance of cell-based assays have been discussed below.
In order to accurately identify the lag, log, and plateau stages of a cell-based assay, you must first become very familiar with the cells being examined and the way they look during every stage of the process. A change in the metabolism of the cell may be identified by any changes that occur in the cell morphology.
A Rauscher erythroleukemia cell line provided an excellent example of these transient, morphological alterations in the cells. It was found that if the flasks containing the cell cultures were moved from the incubator to the microscope, thus jostling the cultures, then transient protrusions would be formed. However, these protrusions would automatically disappear if the flask remained stationary for a little over fifteen minutes, leaving behind spherical cells.
Such changing metabolism of the cells could affect the bioassay. Therefore, for best results, you should introduce the drug being tested to the cultures when they appear normal and do not have any protrusions. However, what can be considered normal will differ with every cell line. Hence, it is important for those performing the experiment to be very familiar with the cells, through microscopic observation and testing.
Some bioassays make use of cultured cells that may require more than a week. Even if the outer wells contain PBS or water, evaporation may occur during this extended time period. This evaporation may change the concentration of various media components, including salt.
However, evaporation can be minimized in long-term cultures by placing the plates inside a hydration chamber which will allow for greater carbon dioxide (CO2) permeation. This, in turn, will trap the water vapor and reduce the extent of evaporation over the period of a week or more. Furthermore, increasing the maximum volume per well to 200uL from 100uL will also help decrease the effect of evaporation, even in the case of overnight evaporations.
It is essential for the adherent cells to be evenly spaced out on the bottom surface during cell death assays or in proliferation. Moving the assay plate to the incubator from the hood creates a circular vortex of the media, causing the cells to accumulate near the outer edges of the well. This leads the center to become empty or 'bald', even as the cells pile one on top of another along the perimeters of the well.
In that case, many of the cells do not get adequate access to the drug, as well as to the other nutrients. This may cause variation in the results and adversely affect the accuracy of the bioassay. The solution is to allow the cells to adhere slightly before the plate is moved to the incubator from the hood. To prevent the media from becoming basic, its Ph level must be carefully observed. A few of the wells should remain under microscopic observation at every step of the bioassay so as to ensure that there are no abnormalities in the culture.
In any cell-based assay that incorporates the development of color or luminescence, a least 20 to 30 minutes must be allowed for substrate development, at an incubation temperature of exactly 37 degree Celsius. This is because a large number of enzymes have the most activity at 37 degree Celsius. In cases that require a shorter incubation time of around ten minutes, room temperature is preferable so as to avoid extensive movement.
Uneven temperature gradients may be created when you are taking a plate in and out of your 37 degree Celsius incubator. This may compromise precision and lead to variation in the bioassay. For instance, there may be variation between plates or even from one day to another on the same plate. However, using room temperature causes sub-maximal activity in the enzymes, as a result of which the enzyme concentration is altered.
When a 4-parameter drug concentration curve is being developed, then each plateau must be anchored quite far away from the inflection point. This will help prevent the curve that is developed from moving from day to day. Moreover, concentrations that describe both the inflection points must be used. Between these two inflection points, the linear portion brings about a precise determination of EC50.
In Conclusion
These are just some of the factors that may affect the performance of cell-based assays, and which scientists and researchers experienced in assay development endeavor to control. Those attempting the development of bioassays should certainly take note of the above-mentioned points for better and more accurate results.
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