In vitro testing of a biotechnological product for ENS patients who need nasal tissue volume restoration

04.05.2021

The experiment of finding a biotechnological product for patients with ENS who need nasal tissue volume restoration continues!
You can read about the previous stages of the research here: stage 1, stage 2.

We have previously suggested that the best material for such a product would be a three-component graft with the following components:
● methylcellulose;
● cell-free lyophilized adipose tissue matrix;
● MSC.
The next important step is to understand precisely how the cells will be distributed in the biotechnology product, whether they will proliferate (grow and divide) there and function normally. These criteria will allow us to evaluate the efficiency and predict the new material's expected therapeutic effect. Realization of these tasks is possible during an experiment in vitro. In vitro is a special technique of experimenting with laboratory-controlled conditions outside a living organism.

In vitro experiment

The three-component graft was populated with MSCs and placed in favorable conditions for cell growth. Next, the cells' condition and behavior were examined by evaluating the populated material under phase contrast and fluorescence microscopes, where we used fluorescent dyes.

Evaluation of cell viability on the graft

The images from the microscope visualize the cells 24 hours after their settlement on the graft. From left to right: phase-contrast microscope, fluorescence microscope image with FDA — a green marker of living cells and PI — a red marker of dead cells. The collage above and below presents images of two different fields of view.
It is clear that MSCs are evenly distributed throughout the material and adhered to the fat matrix's localization. The fraction of dead cells is stable. We can conclude that the cell-free fat matrix serves as a framework for the activity and growth of cells, migration, and normal functioning. 
Methylcellulose, in turn, helps maintain volume and creates 3D conditions for the reproduction of connective tissue analogs.

Conclusions of the stage

The biotechnological product based on MC and a lyophilized cell-free fat matrix is ​​a suitable object for use in future to replace volume defects and reproduction of connective tissue de novo. This product can be administered minimally invasively through an 18G needle. This procedure will be faster and safer than surgery.
The in vitro test was successful, and the next step was in vivo animal studies. In vivo is a type of experiment that takes place in a living organism.

Stage in vivo

Animal experimentation is necessary to test a biotechnological product's safety and effectiveness in a dynamic living system — the body. Besides, we should ensure that the transplanted material remains stable in the recipient's tissues and does not lose much of its volume. The experiment took place in several stages:
1. Rats were injected subcutaneously with a three-component product along the spine. The volume of injections was 1-2 ml.2. The condition of the rats was regularly checked, and blood tests were performed to monitor the development of immune responses.3. Every seven days after introducing the material, the volume of the subcutaneous implant was measured to determine the percentage of losses. Sampling for histological examinations was also performed.
We have already received intermediate results, and they turned out to be very encouraging.Now the experiment is in the final phase, and soon we will be able to share the final results and plans for the future!

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