CN-122004202-A - Serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid

Abstract

The invention relates to the technical field of cell culture, and discloses serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid. The invention discloses serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid which comprises 1000-1500 mmol/L ethylene glycol, 50-150 mmol/L trehalose, 50-150 mmol/L proline and 5-15% human serum albumin injection. The invention further discloses application of the cell freezing solution in freezing cells. The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution disclosed by the invention does not contain dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) components and basic culture medium components, has definite components, is stable in batch and meets the industrial transformation requirements. In addition, the invention can stably maintain the activity and dryness of the cells for a long time when the cells are frozen, and the resuscitated cells can be directly used for clinical application, thereby meeting the safety requirement and the effectiveness requirement of the clinical application.

Inventors

• WANG WENFENG
• XU ZHONGJIN
• ZHANG LINLIN
• CHEN SI
• ZHU ZIYU

Assignees

• 威海紫光优健科技股份有限公司

Dates

Publication Date

20260512

Application Date

20241108

Claims (6)

1. 1. The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is characterized by comprising the following components in percentage by weight: ethylene glycol 1000-1250 mmol/L Trehalose 50-100 mmol/L Proline 50-100 mmol/L 5% -10% Of human serum albumin The balance of the solvent for cell injection.
2. 2. The serum-free and DMSO-free human umbilical mesenchymal stem cell cryopreservation solution according to claim 1, wherein the cell injection solvent is at least one selected from the group consisting of a compound electrolyte injection, a sodium chloride injection, a Normosol-R injection, a dextran 40 glucose injection, a compound dextran 40 injection and a Bomai A injection.
3. 3. The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to claim 2, wherein the pH value of the serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is 7.2-7.6.
4. 4. A preparation method of serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid, which is characterized by mixing the components in the serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid according to any one of claims 1-3 and performing filtering sterilization treatment.
5. 5. A serum-free DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, characterized by comprising the serum-free DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution and human umbilical cord mesenchymal stem cells of any one of claims 1-4.
6. 6. A method for cryopreserving stem cells, comprising the steps of: Mixing the serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to any one of claims 1-5 with human umbilical cord mesenchymal stem cells in a ratio of 1mL to 5×10 6 ～12×10 6 , then treating for 6-12 h under-75 to-80 ℃, and then transferring to liquid nitrogen for storage.

Description

Serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid Technical Field The invention relates to the technical field of cryopreservation liquid preparation, in particular to a preparation method of serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid. Background Human umbilical cord mesenchymal stem cells (UC-MSC) refer to a multifunctional stem cell existing in neonatal umbilical cord tissues, have strong self-renewal capacity, and can be differentiated into a plurality of tissue cells. Umbilical cord mesenchymal stem cells (UC-MSCs) have the advantages of firstly self-renewing, large-scale proliferation, stable passage and rapid cell growth after passage, secondly low immunogenicity, UC-MSCs do not express histocompatibility antigens such as HLA-DR, HLA-DP, HLA-DA and surface co-stimulatory molecules CD80, CD86 and CD40, but express immunosuppressive factors HLA-G, indoleamine 2, 3-peroxidase (IDO), PGE2, IL-10, IL-6, vascular Endothelial Growth Factors (VEGF) and TGF-beta, which enable the UC-MSCs to have low immunogenicity and the capability of inducing immune tolerance, thirdly, the potential of inducing differentiation, and UC-MSCs can be induced into adipocytes, chondrocytes, bone cells, neuronal cells, myocardial cells and the like in corresponding induction culture mediums. In the traditional medical field, umbilical cords are considered waste and are often disposed of at will. However, the International Biotechnology Commission on ISO/TC276 has clearly defined its new value in that by harvesting the umbilical cord and going through a specific manufacturing process, it can be converted to a precious biological resource, which is visually known as a "seed of life" in the life banking. Umbilical cord mesenchymal stem cells have been widely used in various clinical studies on a global scale, and have shown great potential in particular in the treatment of multiple and problematic diseases. Such diseases include, but are not limited to, arthritis, stroke, liver disease, diabetes, cardiovascular disease, and the like. The results of clinical researches show that the umbilical cord mesenchymal stem cells have remarkable curative effects in the aspects of promoting tissue repair, regulating immune response, improving disease symptoms and the like. Fresh cell products are stored for a short period of time, and in order to extend the cell shelf life or preserve the cell products for a long period of time, cell cryopreservation is typically performed, and the cells are preserved in a liquid nitrogen environment at-80 ℃ or-196 ℃. Cell cryopreservation requires a cell cryopreservation protective solution to reduce cell damage. Cryoprotectants are generally classified into two main types, permeable cryoprotectants and impermeable cryoprotectants, depending on whether they penetrate the cell membrane or not. The permeability cryoprotectant is mostly small molecule neutral substances, and mainly comprises glycerol, DMSO, propylene glycol, ethylene glycol, acetamide, methanol and the like. The non-permeability protective agent is mostly macromolecular substances, and mainly comprises trehalose, polyethylene glycol, hydroxyethyl starch, polyvinylpyrrolidone (PVP), dextran, albumin and the like. DMSO is the most commonly used osmotic cryoprotectant, widely used for cryopreservation of cells. At the cellular level, DMSO is also an intracellular factor for DNA teratogenesis, and s=o bonds in the molecule may chemically react with intracellular proteins, causing protein denaturation. Clinically, the use of DMSO-cryopreserved cells causes extensive physical discomfort, most commonly diarrhea (about 50%), severe damage and even death (0.2%) to the nervous system, respiratory system of the patient. Therefore, there is a need for a new serum-free, DMSO-free, wash-free, program-free, temperature-lowering clinical-grade cell cryopreservation solution. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a serum-free and DMSO-free washing-free program-free clinical cell freezing solution. Therefore, the invention provides serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid, and a preparation method and application thereof, wherein the stem cell cryopreservation liquid can avoid toxic and side effects of DMSO in the traditional stem cell cryopreservation liquid on a human body, avoid exogenous viral factor risks caused by serum/animal source protein components in the traditional stem cell cryopreservation liquid, and can avoid the washing operation of washing away the cryopreservation liquid in a clean laboratory before clinical use of the traditional cryopreservation cell, prevent the risk of microbial pollution possibly caused by a washing process, reduce potential safety hazards and simplify the clinical use flow of umbilical cord mesenchymal stem cell products. The inve