In vitro assays for alloreactivity to cells

[neuropath]

Hi,

 

I am quite new to immunology and need some help with designing assays to test the alloreactivity to a particular type of cells in vitro. The goal of my study is to evaluate the suitability of a particular cell type for potential human transplantation. It is a very early study and I have to first show that the cells are not highly immunogenic. I prefer to use in vitro assays rather than animal assays due to resource constraints as well as ethical considerations.

 

From my reading up of the literature, I gathered that there are two common assays to quantify the alloreactivity to a particular cell population - PBMC proliferation assay and T-cell activation assay.

 

T-cell activation is measured by the expression of cytotoxic molecules by T-cells such as granzyme B and perforin. It is normally performed using purified CD3+ pan T-cells.

 

My questions are:

 

1) Is it possible to use whole blood mononuclear cells to do T-cell activation assays? Since whole blood MNCs contain 45-70% CD3+ T-cells, wouldn't it be possible to quantify the number of granzyme B and perforin expressing cells on gated CD3+ T-cell populations?

 

2) What is the minimum number of individual donor PBMCs or T-cells samples that needs to be tested to make the results statistically acceptable?

 

3) Are there any non-cell based assays e.g. PCR-based assays that can be used to predict the alloreactivity of a particular cell population?

 

I would appeciate it greatly is anyone could help shed some light on these questions.

 

Many thanks.

 

-neuropath-