Solid medium for cryopreservation of corneal model, preparation method and application method

Abstract

The invention discloses a solid medium used for cornea model low temperature preservation and a preparation method and an application method thereof. The solid medium is prepared by a basic medium and an agarose aqueous solution according to the volume ratio of 1:1 by steps of uniform mixing and coagulation; the mass concentration of the agarose aqueous solution is 0.5-5%; the basic medium takes a mixed solution of a DMEM nutrient solution and a F12 nutrient solution as a basic solution, fetal calf serum, retinoic acid, human epidermal growth factor, insulin, hydrocortisone, adenine, triiodothyronine, glutamine and a low temperature protective agent are added; the low temperature protective agent is a mixture of HEPES, cane sugar, glycine, alanine, adenosine, vitamin E, vitamin C, hyaluronic acid and chondroitin sulfate. The solid medium has the advantage of reducing low temperature anoxic damage on histocyte, can effectively guarantee the accuracy of the test result of a cornea model, and has the advantages of convenient operation and long-distance transport.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a solid culture medium for low-temperature preservation of a cornea model, a preparation method and a use method. Background technique [0002] With the development of in vitro organ culture technology, the 3D recombinant corneal epithelial model based on in vitro cell culture has shown a unique trend in the field of eye irritation risk assessment. The 3D corneal epithelial model constructed in vitro is similar to the three-dimensional structure of human corneal epithelial cells, which not only can more truly reflect the human body's response to chemicals, but also accurately predict the irritation of chemicals, and has a wider range of types for compound screening. In addition to the development of dosage forms with different efficacy purposes, the scope of application also includes the hazard assessment of specific ingredients in cosmetics. The Organization for...

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Problems solved by technology

However, under low temperature conditions of 4°C to 8°C, especially under a relatively anoxic aseptic storage condition, the balance of the energy supply system is broken, resulting in the blockage of the intracellular and extracellular ion pumps in the cornea model, resulting in intracellular and extracellular The imbalance of Na+ / K+ ratio and Ca2+ concentration will affect the enzymes and biochemical reaction processes that rely on such ions inside the cell, resulting in the obstruction of cell physiological activities, damage to cell membrane and cytoskeleton, and manifested as cell swelling, necrosis or apoptosis; in addition, with As the storage time increases, the cells will also switch from aerobic metabolism to anaerobic metabolism, which will generate a large number of free radicals and acids. Free radicals will selectively attack the cell membrane system, aggravate the damage of cells at low temperature, and eventually cause the skin model to lose its activity.

[0007] Chinese patent 200910078302.X mentions that nutrients and agar are added to the basal culture medium to make a solid medium for preserving tissue-engineered skin, but this medium is limited to storage at room temperature. Due to various factors such as region and season, To maintain normal temperature storage conditions requires special temperature control equipment, which greatly increases the transportation cost; if the medium is used for low temperature storage and transportation, in the long-term low temperature hypoxia state, the way of cell energy utilization will change, from the original aerobic Respiration is converted to glycolysis, the energy utilization rate is greatly reduced, and the metabolic substrates required for glycolysis need to be replenished in time

At the same time, it is necessary to supplement substances that can improve the activity of various biological enzymes. Antioxidants should also be used to remove a large number of acids and free radicals produced by glycolysis. From the perspective of composition, the medium only contains substances that can maintain the normal physiology of tissue engineered skin. The active substances do not have the components that can satisfy the conversion of energy metabolism and inhibit the damage of metabolites, so they do not have the effect of effectively reducing the damage of hypoxia and hypoxia. Vitality declines, organizational structure is destroyed, and follow-up operations cannot be performed

[0008] So far, there is no ideal low-temperature storage and transportation medium for corneal models. This medium should reduce hypoxia damage to tissue cells, effectively ensure the accuracy of corneal model test results, and facilitate operation and long-distance transportation.

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