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CN-121970744-A - Cryopreserved stem cell diluent and application thereof

CN121970744ACN 121970744 ACN121970744 ACN 121970744ACN-121970744-A

Abstract

The application relates to the technical field of biology, in particular to a frozen stem cell diluent and application thereof, wherein the frozen stem cell diluent comprises cysteine and polyvinylpyrrolidone besides a compound electrolyte injection, the concentration of the cysteine in the frozen stem cell diluent is 1-10 mM, and the concentration of the polyvinylpyrrolidone in the frozen stem cell diluent is 0.02-0.1 g/mL. The frozen stem cell diluent can improve cell activity and cell distribution after the frozen cell preparation is recovered, and monitor the application of cells in rat living body imaging.

Inventors

  • ZHANG XIAOPING
  • MAO HAIHUA
  • WANG JINE
  • ZHANG YANGZI

Assignees

  • 上海昂昇生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20260319

Claims (10)

  1. 1. The frozen stem cell diluent is characterized by taking the volume of the frozen stem cell diluent as a reference, and further comprising cysteine and polyvinylpyrrolidone, wherein the concentration of the cysteine in the frozen stem cell diluent is 1-10 mM, and the concentration of the polyvinylpyrrolidone in the frozen stem cell diluent is 0.02-0.1 g/mL.
  2. 2. The lyophilized stem cell diluent according to claim 1, wherein the concentration of cysteine in the lyophilized stem cell diluent is 2-8 mM, preferably the concentration of cysteine in the lyophilized stem cell diluent is 5mM; And/or the concentration of the polyvinylpyrrolidone in the frozen stem cell diluent is 0.03-0.08 g/mL, preferably, the concentration of the polyvinylpyrrolidone in the frozen stem cell diluent is 0.06g/mL.
  3. 3. The cryopreserved stem cell dilution according to claim 1, wherein the stem cells are mesenchymal stem cells, preferably the stem cells are human umbilical cord mesenchymal stem cells; and/or, the frozen stem cell diluent is suitable for frozen stem cell activity and/or distribution detection.
  4. 4. The use of the lyophilized stem cell diluent according to any one of claims 1-3 in stem cell activity and/or distribution detection.
  5. 5. A method for detecting activity and/or distribution of frozen stem cells, which is characterized in that the frozen stem cell diluent as defined in claim 1 or 2 is added into a stem cell suspension, and then the mixture is incubated, injected into animals and subjected to in-vivo imaging detection, wherein the stem cells carry labeled proteins.
  6. 6. The method of claim 5, wherein the stem cell suspension is a cryopreserved resuscitated stem cell suspension; And/or, the stem cell suspension comprises stem cells and frozen stock solution.
  7. 7. The method of claim 5, wherein the volume ratio of stem cell suspension to lyophilized stem cell diluent is (0.5-1.5): 1.5-4.5), preferably wherein the volume ratio of stem cell suspension to lyophilized stem cell diluent is 1:3.
  8. 8. The method according to claim 5, wherein the incubation time is 0.5h to 1.5h, preferably 1h.
  9. 9. The method of claim 5, wherein the stem cells are mesenchymal stem cells, preferably, the stem cells are human umbilical cord mesenchymal stem cells; and/or the marker protein comprises any one or more of Luc, GFP, EBFP, EYFP, mCherry or tdTomato; and/or, injecting a luciferin substrate prior to said detecting.
  10. 10. The method of claim 5, wherein the animal comprises at least one of a rat, a mouse, a nude mouse, or a rabbit; And/or the injection comprises at least one of subcutaneous injection, intradermal injection or cavernous injection, preferably the injection is a cavernous injection, more preferably the injection is a bilateral corpora cavernosa injection.

Description

Cryopreserved stem cell diluent and application thereof Technical Field The application relates to the technical field of biology, in particular to a freezing stem cell diluent and application thereof. Background Diabetic erectile dysfunction (diabetes mellitus-induced erectile dysfunction, DMED) is a common complication for male diabetics, with an overall prevalence of 52.5% in diabetic male patients. Age, diabetes progression, poor glycemic control, hypertension, hyperlipidemia, sedentary lifestyle, smoking and the presence of other diabetic complications have been shown to be associated with DMED. The pathophysiology of diabetic ED is multifactorial, neurogenic, endothelial and/or smooth muscle dysfunction is a major factor, long-term diabetes results in apoptosis and dysfunction of body surface endothelium and smooth muscle, reducing the number of NO-releasing nerves of the penis, resulting in increased oxidative stress of spongiform tissues. Diabetic men have a prevalence of ED 3.5 times that of non-diabetic men. Oral phosphodiesterase-5 inhibitors (PDE-5 i) are the first line treatment of Erectile Dysfunction (ED) and DMED, which include intracavernosal injection and vacuum assist devices. Various treatments have advantages and disadvantages, but in the last decade, no significant progress has been made in the treatment regimen. At present, mesenchymal stem cells are proved to be effective on ED in a plurality of researches, and can restore normal erection function of ED patients, restore internal cavernous pressure of ED model animals/patients, promote blood vessel and cavernous repair, heal ED, and the mechanisms mainly comprise improving vascular injury, restoring smooth muscle, restoring neuron cells, inhibiting inflammatory cytokine production, homing the mesenchymal stem cells, reducing apoptosis of cavernous cells of penis and the like. Mesenchymal stem cells are multipotent stem cells that share the common property of stem cells, namely self-renewal and multipotent differentiation. Mesenchymal Stem Cells (MSCs) have become a new therapeutic approach to treat a variety of diseases in the past few decades, and have been widely used in clinical research. In the early stage, the cell production mechanism carries out fresh cell production and transportation according to the requirement of patients. However, the application of the fresh cells is limited due to the defects of short shelf life, difficult transportation, difficult assurance of consistency among batches, high cost and the like. To address this problem, cell preparation institutions now choose to cryopreserve cell preparations. The frozen cell preparation has the advantages of fixing cells in a specific state, maintaining the original characteristics and functions of the cells and improving the convenience of experiment and clinical application. When clinical cells are injected, the frozen cells are recovered first, and then the recovered cells are injected into a patient, so that the activity and effectiveness of the cell preparation are ensured. Therefore, the cell cryopreservation is of great importance to the stem cell therapy in the disease treatment, so that the storage and transportation problems of fresh stem cells are greatly reduced, and the quality control of cell preparations and the timely treatment of diseases can be better and faster realized. However, short-term and long-term cryopreservation of mesenchymal stem cells may affect cell activity and function, for example, apoptosis may occur in the cryopreserved cells after resuscitation, and immunosuppressive function and multi-directional differentiation potential may be affected, which may also cause trouble to experimental and clinical research and clinical application, and may also make the therapeutic effect of cells significantly impaired. Studies indicate that the immunosuppressive properties of MSCs immediately after thawing are reduced, which is associated with impaired upregulation of indoleamine 2, 3-dioxygenase (IDO) in the face of IFN- γ, and that the multipotent, immunomodulatory and anti-inflammatory properties of adipose mesenchymal stem cells are adversely affected by cryopreservation, but that an adaptation period of 24h is required after cryopreservation cell resuscitation to activate the thawed cells to restore as much as possible impaired stem cell function. From this, it is possible that the decrease in partial functions of MSCs is due to the inability to recover partial functions in a short time after resuscitation of cryopreserved cells. But the frozen cells need to be used in a short time after being resuscitated, and the longer recovery period can cause the problems of reduction of the cell viability and apoptosis, in particular to mesenchymal stem cells. In many fields of application, in vivo imaging also plays a key role in stem cell monitoring. The living body imaging technology is to label genes, cells and living animals by using luciferase, an