阅读说明：本技术 一种DMSO-Free细胞冻存液的设计和制备方法 (Design and preparation method of DMSO-Free cell cryopreservation solution ) 是由 黄火能 于 2021-09-09 设计创作，主要内容包括：本发明公开了一种DMSO-Free细胞冻存液的设计和制备方法,包括以下步骤,S1：选取聚谷氨酸、透明质酸和海藻糖三种化合物作为保护液,分别进行细胞冻存实验；S3：确定成分为化合物聚谷氨酸、化合物透明质酸和化合物海藻糖的一种,按照保护液所占冻存液的配比不同,选出最优配方；S4：将聚谷氨酸*透明质酸、聚谷氨酸*海藻糖、透明质酸*海藻糖和聚谷氨酸*透明质酸*海藻糖等比例混合作为冻存保护液,进行细胞冻存；本发明不依托传统的思路寻找,而是通过已经高度发展的化妆品领域中的成分,发现可以应用在细胞冻存领域的材料,追求接近使用DMSO效果的细胞存活率,但却消除DMSO所附带的毒性,且采用低成本原料,使冻存液可广泛适用于医疗方面。(The invention discloses a design and preparation method of a DMSO-Free cell frozen stock solution, which comprises the following steps of S1: selecting three compounds, namely polyglutamic acid, hyaluronic acid and trehalose as protective solutions, and performing cell cryopreservation experiments respectively; s3: determining one of polyglutamic acid compound, hyaluronic acid compound and trehalose compound as a component, and selecting an optimal formula according to different proportions of the protective solution in the frozen stock solution; s4: mixing polyglutamic acid hyaluronic acid, polyglutamic acid trehalose, hyaluronic acid trehalose and polyglutamic acid hyaluronic acid trehalose in equal proportion to serve as a cryopreservation protection solution, and performing cell cryopreservation; the invention does not depend on the traditional idea for searching, but finds materials which can be applied to the field of cell cryopreservation through the components in the field of cosmetics which are highly developed, pursues the cell survival rate which is close to the effect of using DMSO, but eliminates the toxicity attached by the DMSO, and adopts low-cost raw materials, so that the cryopreservation liquid can be widely applied to the aspect of medical treatment.)

1. A design and preparation method of a DMSO-Free cell frozen stock solution is characterized by comprising the following steps:

s1: selecting three compounds, namely polyglutamic acid, hyaluronic acid and trehalose as protective solutions, and performing cell cryopreservation experiments respectively;

s2: screening out a compound with the effect most similar to that of DMSO, and removing a compound basically without cryopreservation protection capability;

s3: determining one of polyglutamic acid compound, hyaluronic acid compound and trehalose compound as a component, and selecting an optimal formula according to different proportions of the protective solution in the frozen stock solution;

s4: mixing polyglutamic acid hyaluronic acid, polyglutamic acid trehalose, hyaluronic acid trehalose and polyglutamic acid hyaluronic acid trehalose in equal proportion to serve as a cryopreservation protection solution, and performing a cell cryopreservation experiment;

s5: judging the influence of the interaction between the selected protective agents on the cell freezing and storing effect, determining the composition of the protective agents in the freezing and storing liquid to obtain a mixture X, and selecting one of polyglutamic acid, hyaluronic acid and trehalose if the effect is not ideal;

s6: if the component is determined to be the mixture X, performing experiments on the mixture X according to different component proportions and different proportions of the protective solution in the frozen stock solution, and selecting an optimal formula;

s7: and screening out the optimal cryopreservation method suitable for the protective solution by controlling the temperature reduction process and the like to obtain the DMSO-Free cell cryopreservation solution formula.

2. The method for designing and preparing a DMSO-Free cell cryopreservation solution as defined in claim 1, wherein the method comprises the following steps: and in the step S1, heat exchange, the experiment method in the step S4 carries out a plurality of experiments with the proportion of 5% -20%, and the DMSO is frozen and stored with the proportion of 10%.

Technical Field

The invention relates to a design and preparation method, in particular to a design and preparation method of a DMSO-Free cell frozen stock solution, and belongs to the technical field of cell frozen stock solutions.

Background

Stem cell therapy is a very promising approach to the treatment of various diseases, which is currently rapidly developing. The development of the technology requires the construction of a stem cell bank as a support, and the cryopreservation of cells is one of the key technologies. When the cells are frozen, water in the environment inside and outside the cells can form ice crystals, so that a series of physicochemical changes occur in the cells, such as mechanical damage, electrolyte concentration increase, osmotic pressure change, dehydration, pH value change, protein denaturation and the like, and even cell death can be caused in severe cases. Therefore, the cell freezing solution needs to be added with protective agent components such as glycerol, DMSO and the like to lower the freezing point, so that water in the cells can permeate out of the cells before freezing under the condition of slow cooling, and thus, the cell damage caused by the formation of ice crystals can be reduced. The quality of the frozen stock solution has a significant influence on the quality of the cells after the frozen stock.

The currently and generally used cell cryopreservation solution mostly contains dimethyl sulfoxide (DMSO) components, and although DMSO has low cost and good effect, the DMSO can react with protein hydrophobic groups to cause protein denaturation, has certain toxicity to cells, and cannot be used for cryopreservation of cells for treatment. Therefore, the research of a DMSO-Free cell cryopreservation solution with definite chemical components is a hotspot in the field of cell cryopreservation at present, and has high practical application value, although similar commercial products are available abroad, the price is high, the cell cryopreservation solution is difficult to be applied in a cell bank in a large scale, and no mature product is available at home, so that the project aims to search for a nontoxic substitute with similar properties to DMSO from the principle of reducing the formation of ice crystals in cells in a cooling process, and determine the formula of the cell cryopreservation solution by comparing the survival rate of the cells after cryopreservation, thereby developing the cheap and efficient DMSO-Free cell cryopreservation solution for stem cell preservation.

Disclosure of Invention

The invention aims to provide a design and preparation method of a DMSO-Free cell frozen stock solution, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a design and preparation method of DMSO-Free cell frozen stock solution comprises the following steps:

s1: selecting three compounds, namely polyglutamic acid, hyaluronic acid and trehalose as protective solutions, and performing cell cryopreservation experiments respectively;

s2: screening out a compound with the effect most similar to that of DMSO, and removing a compound basically without cryopreservation protection capability;

s3: determining one of polyglutamic acid compound, hyaluronic acid compound and trehalose compound as a component, and selecting an optimal formula according to different proportions of the protective solution in the frozen stock solution;

s4: mixing polyglutamic acid hyaluronic acid, polyglutamic acid trehalose, hyaluronic acid trehalose and polyglutamic acid hyaluronic acid trehalose in equal proportion to serve as a cryopreservation protection solution, and performing cell cryopreservation;

s5: judging the influence of the interaction between the selected protective agents on the cell freezing and storing effect, determining the composition of the protective agents in the freezing and storing liquid to obtain a mixture X, and selecting one of polyglutamic acid, hyaluronic acid and trehalose if the effect is not ideal;

s6: if the component is determined to be the mixture X, performing experiments on the mixture X according to different component proportions and different proportions of the protective solution in the frozen stock solution, and selecting an optimal formula;

s7: and screening out the optimal cryopreservation method suitable for the protective solution by controlling the temperature reduction process and the like to obtain the DMSO-Free cell cryopreservation solution formula.

As a preferred technical solution of the present invention, the experimental method in step S1 performs a plurality of experiments at a ratio of 5% to 20%, and the DMSO is frozen at a ratio of 10%.

Compared with the prior art, the invention has the beneficial effects that:

1. the design and preparation method of the DMSO-Free cell cryopreservation solution is not searched based on the traditional thought, but finds and applies materials in the cell cryopreservation field through the components in the highly developed cosmetic field.

2. The invention relates to a design and preparation method of DMSO-Free cell cryopreservation solution, in particular to a DMSO-Free cell cryopreservation protective solution, which aims to achieve the cell survival rate close to the effect of using DMSO, but eliminates the toxicity attached by the DMSO, and adopts low-cost raw materials, so that the cryopreservation solution can be widely applied to the aspect of medical treatment.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme of a design and preparation method of a DMSO-Free cell frozen stock solution, which comprises the following steps:

a design and preparation method of DMSO-Free cell frozen stock solution comprises the following steps:

s1: selecting three compounds, namely polyglutamic acid, hyaluronic acid and trehalose as protective solutions, and performing cell cryopreservation experiments respectively;

s2: screening out a compound with the effect most similar to that of DMSO, and removing a compound basically without cryopreservation protection capability;

s3: determining one of polyglutamic acid compound, hyaluronic acid compound and trehalose compound as a component, and selecting an optimal formula according to different proportions of the protective solution in the frozen stock solution;

s4: mixing polyglutamic acid hyaluronic acid, polyglutamic acid trehalose, hyaluronic acid trehalose and polyglutamic acid hyaluronic acid trehalose in equal proportion to serve as a cryopreservation protection solution, and performing cell cryopreservation;

s5: judging the influence of the interaction between the selected protective agents on the cell freezing and storing effect, determining the composition of the protective agents in the freezing and storing liquid to obtain a mixture X, and selecting one of polyglutamic acid, hyaluronic acid and trehalose if the effect is not ideal;

s6: if the component is determined to be the mixture X, performing experiments on the mixture X according to different component proportions and different proportions of the protective solution in the frozen stock solution, and selecting an optimal formula;

s7: and screening out the optimal cryopreservation method suitable for the protective solution by controlling the temperature reduction process and the like to obtain the DMSO-Free cell cryopreservation solution formula.

In the step S1, the experimental method is to carry out multiple experiments with the proportion of 5% -20%, and to freeze and store the DMSO with the proportion of 10%.

When the DMSO-Free cell cryopreservation liquid is used specifically, three compounds, namely polyglutamic acid, hyaluronic acid and trehalose, are selected as protective liquids and are respectively used for cell cryopreservation experiments; screening out a compound with the effect most similar to that of DMSO, and removing a compound basically without cryopreservation protection capability; determining one of a compound polyglutamic acid, a compound hyaluronic acid and a compound trehalose, selecting an optimal formula according to different proportions of a protective solution in the frozen stock solution, mixing the polyglutamic acid, the polyglutamic acid trehalose, the hyaluronic acid trehalose and the polyglutamic acid trehalose in equal proportion to serve as the frozen stock protective solution, performing cell freezing, judging the influence of interaction among the selected protective agents on the cell freezing effect, determining the composition of the protective agents in the frozen stock solution to obtain a mixture X, and selecting one of the polyglutamic acid, the hyaluronic acid and the trehalose if the effect is not ideal; if the component is determined to be the mixture X, performing experiments on the mixture X according to different component proportions and different proportions of the protective solution in the frozen stock solution, and selecting an optimal formula; and screening out the optimal cryopreservation method suitable for the protective solution by controlling the temperature reduction process and the like to obtain the DMSO-Free cell cryopreservation solution formula.

In the present invention, unless otherwise explicitly specified or limited, for example, it may be fixedly attached, detachably attached, or integrated; can be mechanically or electrically connected; the terms may be directly connected or indirectly connected through an intermediate, and may be communication between two elements or interaction relationship between two elements, unless otherwise specifically limited, and the specific meaning of the terms in the present invention will be understood by those skilled in the art according to specific situations.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.