SU1751200A1 - Method for fixing primary hepatocytes for extracorporal loop of artificial liver system - Google Patents

Abstract

Uses: biotechnologists. The essence of the invention: primary hepatocytes are fixed directly on the surface of the carrier - foamy, open-cell hydrophilic polyurethane.

Description

The invention relates to biotechnology, in particular, carriers for the cultivation of human and animal cells, and can be used in medicine for creating artificial liver devices.

The problem of carriers for the cultivation of substrate-dependent cells is one of the topical issues in cell biology and biotechnology, while, in addition to bioaffinity and non-toxicity, in some cases the most important characteristic is the development of the surface of the substrate, which allows to create a high ratio of surface cultivation to its scope. Thus, a carrier is known, consisting of a physiologically acceptable mesh of fibers having a porosity of 40-95% and a pore size of 10-100 microns or an open-cell foam structure.

Obtaining and maintaining a short-term primary culture of the liver in vitro is a difficult and not fully accomplished task, since the hepatocytes have increased demands on the culture conditions and, especially, on the carrier to which they are attached. Adhesion of primary hepatocytes on non-horizontal surfaces is particularly difficult due to their high tendency to sedimentation and instability to mechanical effects. There is a way to attach hepatocytes to a commercial dextran-based microcarrier (Cytodex) coated with collagen using an artificial liver in a model device.

However, in such a system, the hepatocytes are located on the outer side of the spherical surface and are easily subjected to mechanical destruction by particles of microcarriers rubbing against each other, and can also be washed away by the flow of the surrounding liquid. The particles themselves create a significant hydrodynamic resistance to the flow of a liquid and thereby severely limit the flow rate of a liquid medium through a column with a microcarrier.

The purpose of the invention is to reduce cell trauma and maintain full-fledged mass exchange of hepatocytes with the washing medium.

The goal is achieved in that according to the method of fixation of primary hepatocytes, including the planting of hepatocytes on the surface of the carrier, an open-cell hydrophilic

polyurethane, directly on the surface of which is fixed hepatocytes.

The essence is to use as a new carrier substrate for primary hepatocytes a biocompatible, non-toxic, technically simple, functionally effective, affordable and cheap polyester polyurea and other drug with an open-cell foam structure. The structure of this matrix is an irregular pore system, separated by thin film partitions, with openings between them of 100-500 microns. Hepatocytes, penetrating through the openings in the septa, are retained within the pores and attached to the walls of the enutri. In this way, a protective effect from external mechanical effects on the cells is achieved, while at the same time their free mass exchange with the surrounding liquid medium is maintained. A significant advantage is the simplicity of the technology for producing such a hepatocyte culture system.

Until now, the use of hydrophilic Tuliester polyurea otnoraporosti polyurethane foam as a carrier for the cultivation of eukaryotic cells, including primary hepatocytes, was not known.

The method is carried out as follows.

Hydrophilic otkaroporisty polyurethane foam is mechanically crushed in an aqueous medium in a fabric shredder to produce irregularly shaped pieces with a transverse size of 3-mm. The polyurethane foam suspension is thoroughly washed with a detergent solution, water, alcohol, sterilized by autoclaving in an aqueous medium and washed with cell culture media, then kept for 12 hours in a nutrient medium containing 10% fetal calf serum.

From the suspension form a column with a ratio of height to its diameter of not less than

3.0-3.5 and remove excess nutrient medium. A suspension of freshly isolated primary hepatocytes (1.5x10 cells per 1 ml) of rat is applied on top of the column and slowly

filtering it through a column (2-3 ml / min), collecting the eluate from below. This operation is repeated twice. As a result, at least 50-60% of the cells are retained on the column.

The viability of cells remaining in the medium after the column was confirmed visually under a microscope, and the number of retained cells was determined by counting the cells in suspension before and after filtering through the column. Under used conditions, 30-40x106 cells were retained in 100 ml of the assembled carrier matrix.

The column is washed with nutrient medium containing 10% fetal bovine serum and left in a CO2 incubator for 18 hours to fix the cells on the carrier.

Control of adhesion of hepatocytes on the carrier was carried out using scanning electron microscopy of samples

Thus, using this polyurethane foam, a high ratio of the area of cultivation of primary hepatocytes to its volume is achieved,

effective protection of cells enclosed inside the pores from external influences, as well as the efficiency of mass transfer between cells and the washing liquid medium.

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Claims (1)

Invention Formula The method of fixation of primary hepatocytes for the extracorporeal contour of the muscular liver, including hepatocytes onto the surface of the carrier, which is characterized by the fact that, in order to reduce the traumatization of the cells and to maintain a full-fledged mass exchange of hepatocytes and the medium, as a carrier use foamy open-cell hydrophilic polyurethane.