CN112772637A - DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution - Google Patents
DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种无DMSO人脐带间充质干细胞注射液冻存液，其特征在于：按体积份计，其制备原料包括复方电解质注射液50～60份，右旋糖苷40葡萄糖注射液20～40份，氯化钠注射液1～10份，葡萄糖注射液1～10份，人血白蛋白30～50份；间充质干细胞无血清培养基1～10份。冻存液中不含DMOS和血清，从而降低临床使用的风险性，避免了血清成分的不确定性及血清培养的不稳定性影响充质干细胞正常的诱导分化功能，本发明冻存液使人脐带间充质干细胞保持良好的冻存效果，人脐带间充质干细胞冻存复苏后具有高的存活率。另外，冻存液冻存的细胞可直接稀释后应用于临床，不需要离心去除冻存液的成分，冻存液可以作为辅料，直接应用于临床给药，更加方便。The invention discloses a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which is characterized in that: in parts by volume, the preparation raw materials include 50-60 parts of compound electrolyte injection, 40 parts of dextran and 20 parts of glucose injection. ~40 parts, 1-10 parts of sodium chloride injection, 1-10 parts of glucose injection, 30-50 parts of human albumin; 1-10 parts of mesenchymal stem cell serum-free medium. The freezing solution does not contain DMOS and serum, thereby reducing the risk of clinical use, avoiding the uncertainty of serum components and the instability of serum culture affecting the normal induction and differentiation function of mesenchymal stem cells. Umbilical cord mesenchymal stem cells maintain a good cryopreservation effect, and human umbilical cord mesenchymal stem cells have a high survival rate after cryopreservation and recovery. In addition, the cells frozen in the cryopreservation solution can be directly diluted and used in clinical practice, without the need for centrifugation to remove the components of the cryopreservation solution.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
Background
Mesenchymal stem cells are multipotent and self-renewing cells, which were first discovered from bone marrow and can differentiate into tissues such as bone, cartilage, fat, muscle, and the like. Mesenchymal stem cells can now be isolated not only from bone marrow but also from tissues such as fat, synovium, umbilical cord, placenta, etc. Compared with bone marrow, the umbilical cord has wide source, convenient material taking, low virus pollution rate (due to the existence of fetal blood barrier), no wound to a donor and no ethical or moral restriction, so that the human umbilical cord mesenchymal stem cells have more ideal development prospect.
The most common technology for freezing and storing the human umbilical cord mesenchymal stem cells is a liquid nitrogen freezing and storing method, and the method is mainly a slow freezing method for freezing and storing the cells by adding a proper amount of protective agent. For example, dimethyl sulfoxide (DMSO) is used as a protective agent. Because if the cells are directly frozen without adding any protective agent, the water inside and outside the cells can quickly form ice crystals, thereby causing a series of adverse reactions. The Chinese patent application CN201510718538.0 discloses a cryopreservation solution and a cryopreservation method for placenta amnion mesenchymal stem cells, wherein the cryopreservation solution consists of DMSO, a basal culture medium and Fetal Bovine Serum (FBS) (10-50%, v/v).
However, because dimethyl sulfoxide has a certain toxic effect, the refrigerating fluid with high content of dimethyl sulfoxide increases the risk of clinical use. In addition, the introduction of exogenous animal serum may increase the risk of foreign protein causing rejection reaction in clinical research and application, and may affect the normal induced differentiation function of mesenchymal stem cells due to the uncertainty of serum components and the instability of serum culture.
Disclosure of Invention
In order to solve the problems, the invention provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following preparation raw materials of 50-60 parts of compound electrolyte injection, 20-40 parts of dextran 40 glucose injection, 1-10 parts of sodium chloride injection, 1-10 parts of glucose injection and 30-50 parts of human serum albumin in parts by volume; 1-10 parts of a mesenchymal stem cell serum-free culture medium.
As a preferable technical scheme, the volume ratio of the compound electrolyte injection to the dextran 40 glucose injection is (1.5-2): 1.
as a preferred technical scheme, the volume ratio of the dextran 40 glucose injection to the human serum albumin is 1: (1.2-1.8).
As a preferred technical scheme, the preparation raw materials of the frozen stock solution of the human umbilical cord mesenchymal stem cell injection without DMSO comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin; 6 parts of a mesenchymal stem cell serum-free culture medium.
The second aspect of the invention provides a preparation method of the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which is characterized by comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
As a preferable technical scheme, the method further comprises the step of placing the prepared frozen stock solution of the DMSO-free human umbilical cord mesenchymal stem cell injection in a medical refrigerator for precooling for more than 30 min.
As a preferable technical scheme, the temperature of the medical refrigerating box is set to be 2-8 ℃.
As a preferable technical scheme, the preparation raw material of the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution also comprises 1-5 parts of serum substitute by volume.
As a preferred technical scheme, the volume ratio of the serum substitute to the mesenchymal stem cell serum-free culture medium is 1: (3-5).
The third aspect of the invention provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which is characterized by comprising the following steps:
s1, taking the frozen stock solution of the DMSO-free human umbilical cord mesenchymal stem cell injection;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
Has the advantages that:
1. the human umbilical cord mesenchymal stem cell injection frozen stock solution does not contain DMSO, but forms high permeability outside cells by taking the dextran 40 glucose injection as an impermeable cell frozen stock protective reagent to dehydrate the cells, thereby protecting the cells from being damaged by the formation of ice crystals and avoiding the adverse effect caused by adding DMSO.
2. The mesenchymal stem cell serum-free culture medium does not contain animal serum or other heterologous proteins, so that the risk of pollution of a cell preparation is reduced. The specific nutritional factors in the serum-free culture medium improve the cell amplification efficiency, maintain the biological characteristics of the stem cells, increase the survival rate of the mesenchymal stem cells and ensure the differentiation capacity of the cells.
3. The cells cryopreserved by the cryopreservation liquid can be directly diluted and applied to clinic without centrifuging to remove components of the cryopreservation liquid, and the cryopreservation liquid can be used as an auxiliary material and directly applied to clinic administration, so that the method is more convenient.
4. The components of the cryopreservation solution can better maintain the biological function of the human umbilical cord mesenchymal stem cells, so that the human umbilical cord mesenchymal stem cells have high survival rate after cryopreservation and recovery, and simultaneously keep better differentiation capability.
Drawings
Fig. 1 is a diagram illustrating an osteogenic differentiation morphology of human umbilical cord mesenchymal stem cells according to example 1 of the present invention;
FIG. 2 is a chondrogenic differentiation morphology of human umbilical cord mesenchymal stem cells in example 1 of the present invention;
FIG. 3 is a diagram showing the adipogenic differentiation morphology of human umbilical cord mesenchymal stem cells in example 1 of the present invention;
Detailed Description
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present disclosure, the definition of the term provided in the present disclosure controls.
As used herein, a feature that does not define a singular or plural form is also intended to include a plural form of the feature unless the context clearly indicates otherwise. It will be further understood that the term "prepared from …," as used herein, is synonymous with "comprising," including, "comprising," "having," "including," and/or "containing," when used in this specification means that the recited composition, step, method, article, or device is present, but does not preclude the presence or addition of one or more other compositions, steps, methods, articles, or devices. Furthermore, the use of "preferred," "preferably," "more preferred," etc., when describing embodiments of the present invention, is meant to refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In order to solve the problems, the first aspect of the invention provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 50-60 parts of compound electrolyte injection, 20-40 parts of dextran 40 glucose injection, 1-10 parts of sodium chloride injection, 1-10 parts of glucose injection and 30-50 parts of human serum albumin in parts by volume; 1-10 parts of a mesenchymal stem cell serum-free culture medium.
The most common technology for freezing and storing the human umbilical cord mesenchymal stem cells is a liquid nitrogen freezing and storing method, and the method is mainly a slow freezing method for freezing and storing the cells by adding a proper amount of protective agent. Because if the cells are directly frozen without adding any protective agent, the water inside and outside the cells can quickly form ice crystals, thereby causing a series of adverse reactions. In the prior art, DMSO is usually used as a protective agent, so that DMSO can permeate into cells to enable water to seep out in a large amount and protect the cells from damage caused by formation of ice crystals, but DMSO has a certain toxic effect and can react with protein hydrophobic groups to cause protein denaturation.
The human umbilical cord mesenchymal stem cell injection frozen stock solution does not contain DMSO, but forms high permeability outside cells by taking the dextran 40 glucose injection as an impermeable cell frozen stock protective reagent to dehydrate the cells, thereby protecting the cells from being damaged by the formation of ice crystals and avoiding the adverse effect caused by adding DMSO.
In order to maintain the cell ion osmotic pressure and the balance of intracellular and extracellular electrolytes, in some preferred embodiments, the volume ratio of the compound electrolyte injection to the dextran 40 glucose injection is (1.5-2): 1.
in order to maintain the cellular colloid osmotic pressure, the volume ratio of the dextran 40 glucose injection to the human serum albumin is 1: (1.2-1.8).
In a preferred embodiment, the preparation raw materials of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin; 6 parts of a mesenchymal stem cell serum-free culture medium.
The human umbilical cord mesenchymal stem cell injection cryopreservation solution provided by the invention does not contain serum, the normal induced differentiation function of the mesenchymal stem cells can be influenced due to the uncertainty of serum components and the instability of serum culture, and particularly, the introduction of exogenous serum can increase the risk of rejection reaction caused by exogenous protein in clinical research and application.
The mesenchymal stem cell serum-free culture medium does not contain animal serum or other heterologous proteins, so that the risk of pollution of a cell preparation is reduced. The specific nutritional factors in the serum-free culture medium improve the cell amplification efficiency, maintain the biological characteristics of the stem cells, increase the survival rate of the mesenchymal stem cells and ensure the differentiation capacity of the cells.
In some preferred embodiments, the raw material for preparing the human umbilical cord mesenchymal stem cell injection frozen stock solution further comprises 1-5 parts of serum substitute, and the determination of the components of the serum substitute is favorable for determining the added content. In some more preferred embodiments, the volume ratio of the serum replacement to the mesenchymal stem cell serum-free medium is 1: (3-5), sufficient nutrients required by carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and the like are provided for the cell freezing and thawing process, and meanwhile, powerful condition guarantee is provided for further proliferation and differentiation of the human umbilical cord mesenchymal stem cells.
The invention provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
When the preparation raw material also comprises a serum substitute, the preparation method comprises the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, and then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection, the mesenchymal stem cell serum-free culture medium and the serum substitute into the receiver, and shaking the mixture while adding the serum substitute until the mixture is uniformly mixed;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
In some preferred embodiments, the preparation method of the human umbilical cord mesenchymal stem cell frozen stock solution further comprises placing the prepared human umbilical cord mesenchymal stem cell frozen stock solution in a medical refrigerator for precooling for more than 30 min.
As a preferable technical scheme, the temperature of the medical refrigerating box is set to be 2-8 ℃.
The third aspect of the invention provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking the human umbilical cord mesenchymal stem cell frozen stock solution;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When the freezing liquid is used, components of the freezing liquid do not need to be removed centrifugally, the freezing liquid can be used as an auxiliary material, and the freezing liquid is taken out and directly diluted to be applied to clinic, so that the freezing liquid is more convenient and faster.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to the examples.
Example 1
Embodiment 1 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin in parts by volume; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of embodiment 1 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the embodiment 1 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Example 2
Embodiment 2 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials of the frozen stock solution comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin by volume; 6 parts of mesenchymal stem cell serum-free culture medium and 2 parts of serum substitute.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the serum replacement was purchased from biotechnology limited, beijing darke.
The second aspect of the embodiment 2 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the embodiment 2 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Example 3
Embodiment 3 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises 55 parts by volume of a compound electrolyte injection, 30 parts by volume of a dextran 40 glucose injection, 5 parts by volume of a sodium chloride injection, 5 parts by volume of a glucose injection, and 40 parts by volume of human serum albumin; 5 parts of mesenchymal stem cell serum-free culture medium and 1 part of serum substitute.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The serum replacement was purchased from biotechnology limited, beijing darke.
The second aspect of the embodiment 3 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the embodiment 3 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Example 4
Embodiment 4 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the preparation raw materials of 50 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin by volume; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
Embodiment 4 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the preparation raw materials of 50 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin by volume; the second aspect of 6 parts of mesenchymal stem cell serum-free culture medium provides a preparation method of DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
Embodiment 4 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the preparation raw materials of 50 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin by volume; the third aspect of 6 parts of the mesenchymal stem cell serum-free culture medium provides a cryopreservation method of the DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation liquid, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Example 5
Embodiment 5 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises 55 parts by volume of a compound electrolyte injection, 30 parts by volume of a dextran 40 glucose injection, 5 parts by volume of a sodium chloride injection, 5 parts by volume of a glucose injection, and 40 parts by volume of human serum albumin; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free culture medium is purchased from Youkang biotechnology (Beijing) limited, and the product number is NC 0103.
The second aspect of the embodiment 5 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the embodiment 5 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Example 6
Embodiment 6 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises 55 parts by volume of compound electrolyte injection, 25 parts by volume of dextran 40 glucose injection, 5 parts by volume of sodium chloride injection, 5 parts by volume of glucose injection, and 40 parts by volume of human serum albumin; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of the embodiment 6 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the embodiment 6 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Comparative example 1
The comparative example 1 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 45 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin in parts by volume; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of comparative example 1 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the comparative example 1 provides a cryopreservation method of a DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Comparative example 2
The comparative example 2 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 55 parts of compound electrolyte injection, 18 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin in parts by volume; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of the comparative example 2 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the comparative example 2 provides a cryopreservation method of the DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Comparative example 3
The comparative example 3 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 20 parts of human serum albumin in parts by volume; 6 parts of a mesenchymal stem cell serum-free culture medium.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of the comparative example 3 provides a preparation method of a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the comparative example 3 provides a cryopreservation method of the DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Comparative example 4
The comparative example 4 provides a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, and the preparation raw materials comprise 55 parts of compound electrolyte injection, 30 parts of dextran 40 glucose injection, 5 parts of sodium chloride injection, 5 parts of glucose injection and 40 parts of human serum albumin in parts by volume; 1 part of mesenchymal stem cell serum-free culture medium and 1 part of serum substitute.
The compound electrolyte injection is purchased from Hebei Tian Chengyao GmbH;
the dextran 40 glucose injection is purchased from Sichuan Konlun pharmaceutical industry Co.Ltd;
the sodium chloride injection is purchased from Tsukamur pharmaceutical Co., Ltd, Guangdong;
the glucose injection is purchased from the national health management corporation;
the human serum albumin is purchased from Oktt enamel AB company;
the mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The second aspect of comparative example 4 provides a method for preparing a DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, comprising the following steps:
s1, adding the dextran 40 glucose injection into a 500mL receiver according to the formula, then slowly adding the compound electrolyte injection, the sodium chloride injection, the glucose injection and the mesenchymal stem cell serum-free culture medium, and shaking while adding until uniformly mixing;
s2, slowly adding human serum albumin with the formula amount, and evenly mixing the mixture in an upside-down inverted manner to obtain the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution.
The third aspect of the comparative example 4 provides a cryopreservation method of the DMSO-free human umbilical cord mesenchymal stem cell injection cryopreservation solution, which comprises the following steps:
s1, taking a human umbilical cord mesenchymal stem cell frozen stock solution which is pre-cooled for 50min in a medical refrigerator at the temperature of 5 ℃;
and S2, resuspending the human umbilical cord mesenchymal stem cells in the cryopreservation liquid, filling the cryopreservation liquid into a cryopreservation bag, cooling by a programmed cooling instrument, and storing the cryopreservation bag in a liquid nitrogen tank.
When in use, the frozen stock solution does not need to be centrifuged to remove components, can be used as an auxiliary material, is taken out and then is directly diluted for clinical application.
Evaluation of Performance
1. Human umbilical cord mesenchymal stem cell survival rate test
The cell survival rate after cryopreservation recovery of the human umbilical cord mesenchymal stem cells in the examples 1-6 and the comparative examples 1-4 is detected, the cell survival rate of more than or equal to 90% is recorded as grade A, the cell survival rate of more than or equal to 85% but less than 90% is recorded as grade B, the cell survival rate of less than 85% is recorded as grade C, and the detection results are shown in table 1.
2. Human umbilical cord mesenchymal stem cell differentiation capacity test
Detecting the differentiation capacity of the human umbilical cord mesenchymal stem cells in the frozen stock solution of the embodiments 1-5, wherein fig. 1 is an osteogenic differentiation morphology map of the human umbilical cord mesenchymal stem cells in the embodiment 1 of the invention; FIG. 2 is a chondrogenic differentiation morphology of human umbilical cord mesenchymal stem cells in example 1 of the present invention; FIG. 3 is a diagram showing the adipogenic differentiation morphology of human umbilical cord mesenchymal stem cells in example 1 of the present invention; the cell differentiation ability of examples 2 to 5 was similar to that of FIGS. 1, 2 and 3.
TABLE 1
According to the embodiments and the comparative examples, the invention provides the DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution, the preparation method and the freezing method thereof, the frozen stock solution does not contain DMOS and serum, so that the risk of clinical use is reduced, and the influence of uncertainty of serum components and instability of serum culture on the normal induced differentiation function of the mesenchymal stem cells is avoided. In addition, the frozen cells of the frozen stock solution can be directly diluted and then applied to clinic without centrifuging to remove the components of the frozen stock solution, and the frozen stock solution can be used as an auxiliary material and directly applied to clinic administration, so that the clinical administration is more convenient.
Finally, it should be understood that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
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