In-vitro screening and culturing method for tissue engineering skin seed epidermal keratinocyte high-simulation in-vivo cell extracellular matrix attachment

Abstract

The invention discloses the culturing, screening and amplification technology for keratinocytes. The bottlenecks are always hot spots and difficulty in biological cell field research. At present, separation and purification are conducted through basilar membrane significant adhesion characteristics, and the keratinocytes are obtained through a three-dimensional culturing and amplification means. The technology is suitable for depending on amplified anchorage-dependent cells and providing a three-dimensional high-simulation in-vivo cell extracellular matrix system for screening and separation of target cell levels. The in-vivo attachment environment is highly simulated, and a novel biological cell culturing technology for culturing adult (embryo skin) skin cells is provided. Adhering, screened and cultured target keratinocytes in the environment of high-simulation substrates have high passage capacity and multiplication capacity and can form a cortex structure under the in-vitro environment. Immunogenicity cells are screened out to safely obtain immune escape, transplanting time is prolonged, tissue engineering clinical application is improved, wound healing is promoted, skin diseases are treated, and a clinical treatment-grade cell solution with absolute advantages is provided.

Description

1. Technical field: [0001] The invention belongs to the field of tissue engineering skin seed keratinocyte (KC) technology and tissue engineering. It relates to an in vitro construction of a highly simulated in vivo attachment environment for separation and screening, and a technology for culturing and expanding skin keratinocytes (KC) cells. The KC cells obtained in the present invention are used as active seed cells in tissue engineering skin construction technology. 2. Background technology: [0002] The skin is the largest organ of the human body. It plays a very important physiological role in resisting biological invasion, ultraviolet radiation, preventing water loss, regulating body temperature and maintaining individual appearance. It is an important part of the human immune system, and skin cells are the composition The basic unit of this organ, the basic research field of skin cells is an important scientific research way to reveal skin health, development, preven...

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Problems solved by technology

Therefore, if the trophoblast is not used, it is possible that the differentiation of skin cells during long-term culture may cause uncontrollable development of subsequent research;

[0017] Method 6 (homologous fibroblast attachment screening method): Scholar Jin Yan et al. used references to improve scholars HagarB and Dunnwald mouse fibroblasts, changed the donor fibroblasts pre-expanded in vitro as the attachment substrate as a trophoblast, and cultured homologous fibroblasts. Skin cells, but the patent does not specify the detailed steps of how to prepare the trophoblast plate, the plating cycle is short, the fibroblast adhesion force is low, and the repeated PBS cleaning and purification process of screening will lose part of the cultured fibroblasts that successfully adhere to the target, thus Reduce the yield of target skin cells. Skin cells are theoretically homogeneous. Another technical problem is that the two cells share the same culture environment. Considering the complexity of the culture medium, the two cells may grow competitively. Proliferation inhibition and other phenomena, the subsequent separation of target cells is technically difficult, and it is not superior to the existing six common methods. It is suggested that this method can be changed into a compound recipe and added to other screening materials with good covering effect and relatively strong adhesion behavior. Increase the yield of target skin cells, or inactivate fibroblast plated cells or add essential factors during culture

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