Question

You want to use retroviruses to deliver DNA to a type of cells to promote certain phenotype. Before you start, you want to estimate the delivery efficiency. The cells are spread on a solid support and covered with an aqueous medium. The culture dish is 6 cm in diameter and the medium is 6 mm deep. You add the virus particle at the top of the medium. The virus has a half life of 8 hours in solution. The diffusion coefficient of the virus is 10^-7 cm²/s.

(a) Estimate the fraction of virus particles that reach cells within their half life when the medium is stagnant. Hints: (1) using scaling analysis to find out the time scale for the virus to diffuse from top to bottom; (2) assume death of the virus follows the kinetics of first order reactions.

(b) Comment on the efficiency of delivery. Suggest approach(es) to improve the delivery efficiency

Answer 1

The process of delivering viral cells into host cells like bacteria is known as transduction. Transduction of bacterial/host cells by viruses can be carried out by plating the viral cells/phage particles onto the host cells, wherein they get adsorbed onto the host cell surface. Transduction can occur naturally or can be carried out artificially in a lab. Artificial methods of transduction is also known as transfection. A fraction of the host cells get infected by the viruses during this incubation period. The number of host cells that have been incubated give us the efficiency of transfection.

The fraction of virus particles adsorbed onto the host cell surface is also represented by the fraction of cells active. This is being denoted by f_active, and is given by the following equation :

Here,

D = 10^-7cm²/s,

L = 6mm = 0.6 cm

V = r²L = 3.14 x 6 x 6 x 0.6 = 67.8 cm³.

t = 8 hours (here the half life is the time for which the experimental observations are being studied)

Since it has been mentioned in the question that the decay of transfecting viruses have taken place in this experiment, therefore the rate of decay should be taken into consideration. As shown in the image, the K_decay value would yield the value of , and help in calculating the value of f_active. Without the value of K_decay or , however, it is not possible to calculate f_active, when viral decay is taking place.

The decay rate of retroviruses can also be calculated using the half-life which has been provided in the question. However, for this formula, knowing the amount of viruses present in the stock solution (in the beginning of the experiment) is essential. If the value of N (viral amount) is provided, then using first order kinetics, the decay rate can be calculated by :

K_decay = (0.693/half life) x N = (0.693/8) x N = 0.08 N.

(Please look for the missing parameters and calculate f_active using the formulae provided).

Transfection efficiency can be calculated by assessment of the fraction of the cells expressing the retroviral markers post the incubation period. It is numerically expressed by

Transfection efficiency () = no. of cells transfected by the virus / total numbe rof cells present.

This reflects the fraction of transfected cells that we calculated earlier. Therefore, higher the fraction, greater the transfection efficiency.

The steps that can be taken to improve transfection efficiency or delivery efficiency are :

• Increase the concentration of the virus to increase the titer.
• Renewal of the media post incubation. After the cells are incubated with the transduction mix, the mix should be discarded and replaced with fresh culture media.
• The cells have to be in good health in order for transduction to be successful. Mycoplasma infections are common in cell cultures, leading to alterations in DNA, RNA and protein levels, as well as cellular metabolism. Therefore checking for mycoplasma contamination is essential in cell cultures.
• Several enhancers are commercially available which help in increasing delivery efficiency. These enhancers inlclude DEAE dextran, polybrene, protamine sulfate.
• Use spinfection to bring about transduction of viruses into the cells. Spinfection implies adding viruses to cells in relatively smaller volumes followed by spinning the plate for better diffusion of the viruses and increased efficiency.
• Use of convective devices, that is, porous membranes in order to bring about flow-through transduction instead of static transduction (as has been described in this question) leads to increase in delivery efficiency. Flow through transduction rates have been shown to be higher than static transduction rates, even in lower volumes of the transducing virus. The use of convective devices and spinfection are known to bring the transducing viruses close to the target cells faster than the conventional methods of transduction.