Carrier-free cell sheet and preparation method and application thereof

Abstract

The invention belongs to the field of tissue engineering, and particularly relates to a carrier-free cell sheet and a preparation method and application thereof. The preparation method of the carrier-free cell sheet comprises the steps of transfecting the suicide gene into a supporting cell; planting the supporting cell with the transfected suicide gene at the bottom of a culture container; planting target cells on the supporting cell; after realizing good fusion between the target cells and establishing intercellular connection, starting the suicide gene in the supporting cell for apoptosis to obtain a carrier-free cell sheet containing pure target cells. By adopting the method provided by the invention, a carrier-free cell sheet with good biological characteristics can be obtained, which is a new breakthrough in establishing a carrier-free cell sheet in the field of tissue engineering; meanwhile, the product also provides a feasible scheme for clinical disease treatment. The principle is scientific and reliable, and the process is simple and flexible.

Description

technical field [0001] The invention belongs to the field of tissue engineering, in particular to a carrier-free cell sheet and its preparation method and application. Background technique [0002] Compared with cell sheet products containing scaffold materials (collagen gel, fibrin gel or biofilm), the construction and use of carrier-free cell sheets has the following advantages: ①It can efficiently collect complete and continuous high Density cell sheets can evenly establish two-dimensional and three-dimensional structures of tissues or organs; ②The base of the carrier-free cell sheet has good adhesion to the extracellular matrix, and is easy to transplant into other culture containers in vitro or on the surface of tissues in the body. Suture can be directly adhered to the injured tissue; ③ Since no scaffold material is needed, side effects such as inflammatory response and tissue fibrosis induced by scaffold degradation after implantation in vivo can be avoided. ④ It has...

AI Technical Summary

Problems solved by technology

Among them: ①The temperature-sensitive culture dish method refers to the use of temperature-sensitive materials as cell culture dishes, and the solid-liquid conversion of the culture dish is achieved by changing the temperature, so that the cell sheet is separated from the culture dish. The advantage of this method is that it can be achieved by controlling simple conditions. Separation of the cell sheet from the culture container, the disadvantage of this method is that there may be residual organic matter in the cell sheet, and it is difficult to remove the cell sheet from the culture dish

②The gel method is to plant cells on the gel, then use tweezers to tear the gel out of the culture container, and then digest the gel with protease to obtain the cell sheet. This method is simple to operate, but the disadvantage is the synthesis The gel process is complex and can cause damage to cells during protease digestion

③The magnetic method is to prepare a magnetic cationic liposome MCLS, combine MCLS and RGD to form a colloidal coating, apply this coating on the bottom of the culture well, and place a magnet on the bottom of the culture well, plant the cells in the culture well, wait for After the cells are fused, the magnet is removed, and the fused cells begin to separate from the culture plate gradually from the periphery to form a cell sheet. The advantage of this method is that it is easy to transplant and can reduce the mechanical damage during the transplantation process. Magnetic substance, the toxic effect on the host is unknown

④The polyelectrolyte method forms a polyelectrolyte film, and after the cells are fused on the film, the film is dissolved by applying a voltage to obtain a cell sheet. This method is easy to operate, and the cells retain good mechanical properties and adhesion; The disadvantage is that the polyelectrolyte film cannot provide help and support for the biological activity of the target cells

⑤ The rough particle method is to use rough particles to form a rough monolayer. After the cells are fused on the monolayer, the cell sheet will fall off by blowing and beating. This method does not require enzymatic digestion, which reduces the damage to the cells. A relatively uniform cell sheet is obtained, however, the coarse particles used in this method are complicated to prepare and not easy to operate

For this kind of poorly differentiated cells with higher culture requirements, although the current various methods can also prepare cell sheet products with 3 to 5 layers, because these poorly differentiated cells are separated from the physiological microenvironment in vivo and lack corresponding Supported by the paracrine function of supporting cells, poorly differentiated target cells often differentiate rapidly, changing from a poorly differentiated state of cells to a terminally differentiated state. The original biological characteristics of the target cells are difficult to maintain, which reduces the theory of cell sheet products treatment effect