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The Lentivirus System

#### A Guide to Lentivirus Production (Protocol, Tips, & more!)

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What is Multiplicity of Infection (MOI)?

Summary Video

So you’ve packaged your DNA into a virus and you’re ready to infect your cells! But, how many viral particles are required for 100% infection? In this blog post and video, we’ll explain the concept of Multiplicity of Infection or MOI and take you through how to determine the best MOI for your experiment.

What is multiplicity of infection?

In a nutshell, MOI is the ratio of infectious agents to infection targets. In many cases, it is the ratio of viral particles to target cells in a defined space, such as a cell culture well.

$$MOI = {Infectious \ Agents \over Infection \ Targets} = {Viral \ Particles \over Target \ Cells}$$

For example, if you add 10 million viruses to 1 million cells, you’d have an MOI of 10 and an average probability of 10 viral particles infecting one cell.

$$MOI = {Viral \ Particles \over Target \ Cells}$$ $$MOI = {10 \ million \ viral \ particles \over 1 \ million \ cells}$$ $$MOI = 10$$
A simple sample calculation

Let’s do a quick example calculation! Let’s say you’d like to achieve an MOI of 10. If your virus titer is 1 x 10 6 IU/ml and you are delivering to 1 x 10 5 cells, what volume of virus will you need for your project?

We’ll use the following calculation:

$$MOI = {Virus \ Titer \times Virus \ Volume \over Total \ Cell \ Number}$$ $$MOI = {1 \times 10^6 \ IU/ml \ \times \ ? \over 1 \times 10^5 \ cells}$$ $$? = 1 \ ml$$

So you will need 1.0 ml of virus to achieve an MOI of 10.

Factors that can affect your MOI

So, based on the simple definition of MOI, you would expect that if your MOI was one, then each cell would be infected by one virus.

But the reality is not as simple! Why? Imagine yourself throwing 100 tennis balls into a room that has 100 buckets. Theoretically, there is one ball for every bucket. But in reality, the chance of every bucket getting 1 ball is very low! There are other factors to consider, such as do the buckets have backboards that would make it easier to make the shot?

Similarly, there are factors that can affect how easily viruses can infect their target cells, such as:

• the current state of your cell line: dividing or non-dividing
• the characteristics of the virus: lentivirus, adenovirus, etc.
• the transduction efficiency
• your application: transducing a packaging cell line for virus production, generating a stable cell line for protein production, etc.

For example, if the cell is in a non-dividing state, a higher MOI may be needed to achieve optimal transduction efficiency. This is the case when infecting neuronal cells such as SH-SY5Y with lentiviruses for gene delivery where a much higher MOI of 10-50 can be required.

On the other hand, when it comes to infecting insect cells such as Sf9 cells with baculovirus for viral production, a low MOI of <1 is typically recommended. This is because passaging baculoviruses at higher MOIs increases the possibility of transferring large amounts of defective, non-infectious viruses.

AAV, in contrast, requires MOI ranges of 10,000 to 500,000.

Essentially, if the MOI is too high, it can cause cytotoxicity or other undesired effects. If the MOI is too low, it will not achieve 100% infection.

Do a pilot experiment to find your optimal MOI

So, how do you determine the optimal MOI for your experiment? Simply perform a pilot experiment using a reporter virus on your target cell line!

General workflow:

Step 1: Select 6 MOI conditions to test

For example, using a GFP Lentivirus, design a range of MOIs to use, let’s say, 6 conditions ranging from MOIs 1, 2, 5, 10, 15, and 30. It is typically better to test a lower MOI range to avoid cytotoxicity at the higher MOIs. A good starting point would be to reference commonly used MOI for cancer cells and devise the range around the suggested MOI.

Table 1: A general guide for Lentivirus MOIs of popular cell line models. Adapted from Molecular Therapy (2004) 9, S281

 Cell Line Name Description Suggested MOI A431 Human Epidermal Carcinoma 5 A549 Human Lung Carcinoma 5 B-16F10 Mouse Skin Melanoma, Metastatic 5 BxPC3- Human Pancreatic Adenocarcinoma 10 H3255 Human Non-small Cell Lung Cancer 10 HCT116 Human Colon Carcinoma 5 HeLa Human Cervical Carcinoma 3 Hepa6-1 Mouse Liver Carcinoma 3 HT29- Human Colon Adenocarcinoma 3 Jurkat Human Acute T Cell Leukemia 10 LLC1- Mouse Lung Carcinoma 6 LNCaP Human Prostate Carcinoma 5 MM200 Human Skin Melanoma 5 MCF7- Human Breast Adenocarcinoma 2 MDA-MB231- Human Breast Adenocarcinoma 1 MM-AN Human Skin Melanoma, Metastatic 16 MMC Mouse Breast Carcinoma 4 MRC5- Human Embryonic Lung Fibroblasts 1 NB4 Human Acute Promyelocytic Leukemia 10 PC12 Rat Adrenal Gland Pheochromocytoma 20 SKOV3- Human Ovary Adenocarcinoma 15 U2-OS Human Bone Osteosarcoma 5

Step 2: Infect your target cells and record results

After you have infected your cells, allow the appropriate time to pass before evaluating the fluorescence. For lentiviruses, this is generally 48 -72 hours post infection. Next, record the fluorescence at the various MOIs to determine your transduced cell percentage.

Step 3: Select the minimum MOI at which all the cells are expressing the transgene.

Here is an example of how your results may look like. In this case, a minimum MOI of 10 is required for 100% infection of the target cells.

If your cells are naturally harder to transduce, there are transduction enhancers such as polybrene or our ViralMax Transduction Enhancers to increase infectivity performance.

We hope you found this blog post helpful in determining the optimal conditions for transduction of your virus to your target cells. At abm, we offer a ready-to-use collection of Lenti-, AAV, Adeno, and Retroviruses for any human, mouse, and rat gene! Browse our collection of tools and resources for gene expression today!