Cryopreserved stem cell diluent and application thereof

Abstract

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

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a cryopreservation stem cell diluent and its application. Background Technology

[0002] Diabetic mellitus-induced erectile dysfunction (DMED) is a common complication in men with diabetes, with an overall prevalence of 52.5%. Increased age, duration of diabetes, 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, with neurogenic, endothelial, and / or smooth muscle dysfunction being the main factors. Long-term diabetes leads to apoptosis and dysfunction of the epithelial and smooth muscle, reducing the number of NO-releasing nerves in the penis and increasing oxidative stress in the cavernous tissue. The prevalence of ED in diabetic men is 3.5 times higher than in non-diabetic men.

[0003] Oral phosphodiesterase-5 inhibitors (PDE-5i) are first-line treatments for erectile dysfunction (ED) and diabetic dysplasia of the penis (DMED). Second-line treatments include intracavernosal injection and vacuum-assisted devices. Each treatment method has its advantages and disadvantages, but no major advancements have been made in treatment options over the past decade. Currently, mesenchymal stem cells have been shown to be effective for ED in multiple studies, restoring normal erectile function in ED patients, restoring intracavernosal pressure in ED model animals / patients, promoting vascular and corpus cavernosum repair, and potentially curing ED. The mechanisms primarily include improving vascular damage, restoring smooth muscle, restoring neuronal cells, inhibiting the production of inflammatory cytokines, homing mesenchymal stem cells, and reducing penile cavernous body cell apoptosis.

[0004] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell, possessing the common characteristics of stem cells, namely self-renewal and multi-lineage differentiation capabilities. Over the past few decades, MSCs have emerged as a novel treatment method for various diseases and have been widely used in clinical research. Initially, cell production institutions produced and transported fresh cells based on patient needs. However, the short shelf life, transportation difficulties, batch-to-batch consistency challenges, and high costs of fresh cells limited their application. To address this issue, cell preparation institutions now opt for cryopreserved cell preparations. Cryopreserved cell preparations offer advantages such as fixing cells in a specific state, preserving their original characteristics and functions, and improving the convenience of experimental and clinical applications. In clinical cell injection, cryopreserved cells are first thawed before being injected into the patient, ensuring the activity and effectiveness of the cell preparation. Therefore, cell cryopreservation is crucial for stem cell therapy in disease treatment, significantly reducing the storage and transportation problems associated with fresh stem cells, and enabling better and faster quality control of cell preparations and timely disease treatment.

[0005] However, short-term and long-term cryopreservation of mesenchymal stem cells (MSCs) may affect cell viability and function. For example, after thawing, cryopreserved cells may undergo apoptosis, and their immunosuppressive function and multi-lineage differentiation potential may also be affected. This poses challenges to experimental and clinical research and applications, significantly reducing the therapeutic efficacy of the cells. Studies have shown that the immunosuppressive properties of MSCs are reduced immediately after thawing, which is related to impaired upregulation of indoleamine 2,3-dioxygenase (IDO) in the presence of IFN-γ. The multi-lineage differentiation capacity, immunomodulatory function, and anti-inflammatory properties of adipose-derived mesenchymal stem cells are adversely affected by cryopreservation. However, cryopreserved cells require a 24-hour adaptation period after thawing to activate them and restore their damaged stem cell functions as much as possible. Therefore, the reduction in some functions of MSCs may be because some functions cannot be restored quickly after thawing. However, cryopreserved cells need to be used within a short period after thawing; a prolonged recovery period can lead to decreased cell viability and apoptosis, especially in mesenchymal stem cells.

[0006] Among its many applications, in vivo imaging plays a crucial role in stem cell monitoring. In vivo imaging technology uses luciferase to label genes, cells, and living animals. The luciferase protein expressed by the luciferase gene interacts with the substrate luciferin under oxygen and magnesium conditions. 2+In the presence of ATP, an oxidation reaction occurs, converting some chemical energy into light energy. These light signals are captured by imaging equipment, allowing for the location, survival, proliferation, and distribution of cells expressing luciferase within an animal. However, the reduced cell viability after thawing of cryopreserved mesenchymal stem cells and incomplete activation of luciferase activity affect the distribution of mesenchymal stem cells in in vivo animal imaging, leading to decreased or delayed fluorescence intensity.

[0007] Therefore, there is an urgent need for a solution that can improve the cell viability of cryopreserved mesenchymal stem cells after thawing while maintaining the immunofluorescence activity for detecting stem cell distribution. Summary of the Invention

[0008] To address the aforementioned technical problems, this application provides a method for improving the activity and immunofluorescence activity detection of cell distribution after cryopreserved mesenchymal stem cells are thawed, which can enhance cell viability and distribution after thaw of cryopreserved cell preparations and monitor the application of cells in rat in vivo imaging.

[0009] This application provides a cryopreservation stem cell diluent. Based on the volume of the cryopreservation stem cell diluent, the cryopreservation stem cell diluent includes, in addition to compound electrolyte injection, cysteine ​​and polyvinylpyrrolidone. The concentration of cysteine ​​in the cryopreservation stem cell diluent is 1-10 mM, and the concentration of polyvinylpyrrolidone in the cryopreservation stem cell diluent is 0.02-0.1 g / mL.

[0010] This application also provides the application of the above-mentioned cryopreserved stem cell diluent in the detection of stem cell activity and / or distribution.

[0011] This application also provides a method for detecting the activity and / or distribution of cryopreserved stem cells, which involves adding the above-mentioned cryopreserved stem cell diluent to a stem cell suspension, incubating, injecting into an animal, and performing in vivo imaging detection; wherein the stem cells carry marker proteins.

[0012] The purpose of this application is to provide a method for detecting cell distribution after the thawing of cryopreserved human umbilical cord mesenchymal stem cells, so as to better assess the cell distribution after human umbilical cord mesenchymal stem cells are transplanted into the corpora cavernosa of the animal penis.

[0013] The principle of the application is to prepare a transfected human umbilical cord mesenchymal stem cell and cryopreserve it. Afterwards, the cell cells are taken out and thawed in a 39°C water bath to obtain a cell suspension. A mixed diluent is then added to this cell suspension to prepare a cell preparation. After a certain period of incubation, using male immunodeficient rats as experimental animals, the cell preparation is injected into the corpora cavernosa of the penis of immunodeficient rats, and the cell distribution in the immunodeficient rats is monitored.

[0014] To achieve the above objectives, this application employs the following experimental steps: Preparation of hUC-MSCs stably expressing Luc-GFP: The hUC-MSCs cell preparation frozen in liquid nitrogen was removed and thawed in a 39°C water bath. After thawing, the cryopreservation solution was removed by centrifugation, and an appropriate amount of serum-free culture medium was added to prepare a cell suspension. One day before virus transfection (day 0), hUC-MSCs were seeded in 6-well plates at 4 × 10⁶ cells / well. 5 Cells were inoculated with an appropriate amount of serum-free medium and incubated overnight at 37°C with 5% CO2. On day 1 (transfection day), when the cells reached 30%-50% confluence, the transfection experiment was prepared. Cells were removed from the incubator, the original culture medium was aspirated, and then preheated fresh serum-free medium mixed with polybrene was added. Lentiviral solution was then added to the wells of the experimental group, and the culture plate was gently shaken back and forth. The cells with added virus were then returned to the 37°C, 5% CO2 incubator for incubation. On day 2, the virus-containing medium was removed and replaced with fresh serum-free medium, and the cells were incubated at 37°C with 5% CO2 for 6-8 hours. Cells were then removed from the incubator, the virus-containing culture medium was aspirated, PBS buffer was added, and the culture plate was shaken back and forth to wash the cells with PBS buffer. The PBS buffer was then slowly aspirated, and fresh culture medium was added to the washed cells. The cells were returned to a 37°C, 5% CO2 incubator for overnight culture. GFP fluorescence expression in the cells was then observed daily under an inverted fluorescence microscope. The transfected hUC-MSCs were screened using puromycin, and finally, GFP / Luc stably transfected hUC-MSCs (referred to as T-hUC-MSCs) were selected. Transfection efficiency was detected by flow cytometry; only cells with satisfactory transfection efficiency were used for subsequent experiments.

[0015] Cryopreservation of transfected human umbilical cord mesenchymal stem cells (T-hUC-MSCs): After the T-hUC-MSCs were prepared, they were filled into cryopreservation tubes at a rate of 0.5 mL / tube, then cooled using a programmed temperature-controlled freezer and transferred to a liquid nitrogen tank for cryopreservation.

[0016] The cryopreserved cells were divided into three portions. On the first day, one portion of cells was thawed and placed in a 39 °C water bath. The thawed T-hUC-MSCs were transferred to a clean bench, centrifuged to remove the cryopreservation solution, and then an appropriate amount of serum-free mesenchymal stem cell culture medium was added. The cells were then transferred to a culture flask and incubated in a cell culture incubator for 24 h. After incubation, the cells were digested with trypsin and resuspended for later use. Cell viability was detected using a cell counter and the cells were recorded as fresh cells (FT-hUC-MSCs).

[0017] The next day, two more cell lines were revived and placed in a 39°C water bath. One revived T-hUC-MSCs sample was directly added to 1.5 mL of compound electrolyte injection solution and incubated in a cell culture incubator; this was designated as cryopreserved cells (C-hUC-MSCs). The other revived T-hUC-MSCs sample was added to 1.5 mL of mixed dilution buffer; this was designated as cryopreserved cells + dilution mixture (C-hUC-MSCs + CP). Both T-hUC-MSCs and C-hUC-MSCs + CP were then incubated for 1 hour. Cell viability was assessed using a cell counter after incubation.

[0018] Experimental animals: Immunodeficient rats: 15 SPF-grade male Nude rats that passed quarantine, aged 8-11 weeks, were divided into 3 groups: fresh cell (FT-hUC-MSCs) group (animal numbers 1101-1105), cryopreserved cell (C-hUC-MSCs) group (animal numbers 2101-2105), and cryopreserved cell + dilution mixture (C-hUC-MSCs+CP) group (animal numbers 3101-3105).

[0019] Injection of fluorescein substrate: Weigh 648 mg of fluorescein substrate into a sterile centrifuge tube, dissolve it in 16 mL of physiological saline to prepare a 40 mg / mL substrate solution for later use.

[0020] Substrate preparation was performed in a clean bench under aseptic conditions. 10-15 minutes before the experiment, sodium fluorescein was injected intraperitoneally at a dose of 150 mg / kg body weight. Imaging was performed after the substrate had diffused throughout the body.

[0021] Rats were administered the drug via isoflurane gas anesthesia and a single bilateral injection into the corpora cavernosa of the penis. The animals were not fed before administration and were allowed to drink water normally.

[0022] Injection site: After cell incubation, the cells were injected into both sides of the corpus cavernosum of the penis of immunodeficient rats.

[0023] Administration volume: 50 μL was administered to each side of the corpora cavernosa, for a total of 0.1 mL per animal.

[0024] The injected cell concentration was 9 × 10⁻⁶ 6 cells / mL.

[0025] After administration, carefully observe clinical symptoms (animal appearance, behavior, and response to stimuli) and mortality (time of death or time of discovery of death and pre-mortem reactions, etc.). Observe once a day during the trial.

[0026] Pharmacokinetic distribution assay: After administration to animals, the bioluminescence intensity and tissue distribution of the test substance in the animals were observed using an in vivo imaging system. All animals underwent in vivo imaging monitoring before administration and at 2h, 8h, 24h, 48h, 72h, 120h, 168h, 216h, and 288h after administration. All animals were euthanized when the monitoring signal approached background levels.

[0027] The detection method is as follows: 200 mg / kg of fluorescein substrate was injected via the tail vein. The substrate concentration was prepared at 40 mg / mL, i.e., 1.35 mL was injected into each animal. Immediately after injection, anesthesia was induced with 2.5%-4.5% isoflurane and maintained with 1.5%-3.5% isoflurane. The fluorescence intensity and tissue distribution of the test substance in the animal were observed and photographed using a small animal in vivo imaging system. The detection was completed 5±1 min after substrate injection.

[0028] Animal euthanasia method: Euthanasia is performed using carbon dioxide inhalation. Animal carcasses and remains are disposed of harmlessly by a qualified company arranged by the Laboratory Animal Department.

[0029] Process principle: This application prepares a dilution mixture that has antioxidant properties, inhibits the generation of ROS, reduces oxidative damage to cells, and promotes the generation of ATP. The generation of ATP is the energy required for the activation of luciferase activity, which can activate immunofluorescence activity. At the same time, the dilution mixture also has good biocompatibility, regulates the osmotic pressure inside and outside the cell, and has a protective effect to reduce the damage to cells caused by cryopreservation.

[0030] This application uses male animals as donors, resuscitates cryopreserved and transfected human umbilical cord mesenchymal stem cells, treats them with a dilution mixture, and then transplants them into the bilateral penile corpora cavernosa of rats. In vivo imaging technology is used to detect the distribution of human umbilical cord mesenchymal stem cells in the penile corpora cavernosa of male rats and their application in rat in vivo imaging.

[0031] The beneficial effects of this application include, but are not limited to, the advantages of the cryopreservation stem cell diluent as follows: This cryopreservation stem cell diluent contains non-permeable protectants and antioxidants, which reduce cell damage caused by the cryopreservation solution. At the same time, it can inhibit the production of ROS, reduce oxidative stress damage to cells, and improve the cell viability of human umbilical cord mesenchymal stem cells.

[0032] The luciferase protein expressed by the luciferase gene and its substrate luciferin require oxygen, Mg2+, and ATP energy. Compared to fresh cells, the viability of cryopreserved cells decreases after thawing, and the activity of transfected luciferase is temporarily delayed in recovery due to the low temperature caused by cryopreservation. The addition of the cryopreserved stem cell diluent in this application can promote the activation of luciferase activity, thereby increasing cell viability and luciferase activity in a short period of time after cryopreserved cell thawing, resulting in cryopreserved cells having cell viability similar to that of fresh cells.

[0033] In terms of clinical research and application, it reduces the storage and transportation problems associated with fresh cells, improves the issue of reduced viability of frozen cell preparations during clinical injection, and greatly enhances the convenience of using frozen cell preparations, thus facilitating their application in clinical research and clinical translation. Attached Figure Description

[0034] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 Transfection efficiency of human umbilical cord mesenchymal stem cells.

[0035] Figure 2 It is the viability of human umbilical cord mesenchymal stem cells.

[0036] Figure 3 This is a bioluminescence intensity graph of FT-hUC-MSCs injected into the corpus cavernosum of the penis of rats.

[0037] Figure 4 This is a bioluminescence intensity graph of the FT-hUC-MSCs group rats at different time points after injection into the corpus cavernosum.

[0038] Figure 5 This is a bioluminescence intensity graph of CT-hUC-MSCs injected into the corpus cavernosum of the penis of rats.

[0039] Figure 6 This is a bioluminescence intensity graph of the CT-hUC-MSCs group rats at different time points after injection into the corpus cavernosum.

[0040] Figure 7 This is a bioluminescence intensity diagram of CT-hUC-MSCs+CP group rats injected into the corpus cavernosum.

[0041] Figure 8 This is a bioluminescence intensity graph of the CT-hUC-MSCs+CP group rats at different time points after injection into the corpus cavernosum. Detailed Implementation

[0042] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0043] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0044] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0045] This application provides a cryopreservation stem cell diluent. Based on the volume of the cryopreservation stem cell diluent, the cryopreservation stem cell diluent includes, in addition to compound electrolyte injection, cysteine ​​and polyvinylpyrrolidone. The concentration of cysteine ​​in the cryopreservation stem cell diluent is 1-10 mM, and the concentration of polyvinylpyrrolidone in the cryopreservation stem cell diluent is 0.02-0.1 g / mL.

[0046] In some embodiments, the concentration of cysteine ​​in the cryopreserved stem cell diluent can be 2–8 mM. In some embodiments, the concentration of cysteine ​​in the cryopreserved stem cell diluent can be 3–7 mM. In some embodiments, the concentration of cysteine ​​in the cryopreserved stem cell diluent can be 4–6 mM. In some embodiments, preferably, the concentration of cysteine ​​in the cryopreserved stem cell diluent can be 5 mM.

[0047] In some embodiments, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent can be 0.03–0.08 g / mL. In some embodiments, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent can be 0.04–0.07 g / mL. In some embodiments, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent can be 0.05–0.06 g / mL. In some embodiments, preferably, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent can be 0.06 g / mL.

[0048] In some embodiments, the stem cells may be mesenchymal stem cells. In some embodiments, preferably, the stem cells may be human umbilical cord mesenchymal stem cells.

[0049] In some embodiments, the cryopreserved stem cell diluent can be used for detecting the activity and / or distribution of cryopreserved stem cells.

[0050] This application also provides the application of the above-mentioned cryopreserved stem cell diluent in the detection of stem cell activity and / or distribution.

[0051] In some embodiments, the distribution may be an in vivo distribution.

[0052] This application also provides a method for detecting the activity and / or distribution of cryopreserved stem cells, which involves adding the above-mentioned cryopreserved stem cell diluent to a stem cell suspension, incubating, injecting into an animal, and performing in vivo imaging detection; wherein the stem cells carry marker proteins.

[0053] In some embodiments, the stem cell suspension may be a frozen and thawed stem cell suspension; In some embodiments, the stem cell suspension may include stem cells and a cryopreservation solution. In some embodiments, the cryopreservation solution may include DMSO, HSA, and a compound electrolyte injection solution.

[0054] In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (0.5–1.5):(1.5–4.5). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (0.6–1.4):(1.75–4.25). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (0.7–1.3):(2.0–4.0). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (0.8–1.2):(2.25–3.75). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (0.9–1.1):(2.5–3.5). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (1.0–1.1):(2.75–3.25). In some embodiments, the volume ratio of the stem cell suspension to the cryopreserved stem cell diluent can be (1.0–1.1):(3.0–3.25).

[0055] In some embodiments, preferably, the volume ratio of the stem cell suspension to the cryopreserved stem cell dilution solution can be 1:3.

[0056] In some embodiments, the incubation time can be 0.5h to 1.5h. For example, the incubation time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, or 1.5h. In some embodiments, preferably, the incubation time can be 1h.

[0057] In some embodiments, the stem cells may be mesenchymal stem cells. In some embodiments, preferably, the stem cells may be human umbilical cord mesenchymal stem cells.

[0058] In some embodiments, the marker protein may include any one or more of Luc, GFP, EBFP, EYFP, mCherry, or tdTomato.

[0059] In some embodiments, a fluorescein substrate may be injected prior to the detection.

[0060] In some embodiments, the animal includes at least one of a rat, mouse, nude mouse, or rabbit.

[0061] In some embodiments, the injection may include at least one of subcutaneous injection, intradermal injection, or corpus cavernosum injection. Preferably, in some embodiments, the injection may be a corpus cavernosum injection. More preferably, in some embodiments, the injection may be a bilateral corpus cavernosum injection.

[0062] In some embodiments, the method for detecting the activity and / or distribution of cryopreserved stem cells does not require culturing the cells for 24-72 hours after thawing, but instead involves direct injection into the animal.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0064] Example 1 - Construction of hUC-MSCs stably expressing Luc-GFP The hUC-MSCs cell preparation stored in liquid nitrogen was removed and thawed in a 39°C water bath. After thawing, the cryopreservation solution was removed by centrifugation, and an appropriate amount of serum-free culture medium was added to prepare a cell suspension. One day before virus transfection (day 0), hUC-MSCs were seeded in 6-well plates at 4 × 10⁶ cells / well. 5 Cells were inoculated with an appropriate amount of serum-free medium and incubated overnight at 37°C with 5% CO2. On day 1 (transfection day), when the cells reached 30%-50% confluence, the transfection experiment was prepared. Cells were removed from the incubator, the original culture medium was aspirated, and then preheated fresh serum-free medium mixed with polybrene was added. Lentiviral solution was then added to the wells of the experimental group, and the culture plate was gently shaken back and forth. The cells with added virus were then returned to the 37°C, 5% CO2 incubator for incubation. On day 2, the virus-containing medium was removed and replaced with fresh serum-free medium, and the cells were incubated at 37°C with 5% CO2 for 6-8 hours. Cells were then removed from the incubator, the virus-containing culture medium was aspirated, PBS buffer was added, and the culture plate was shaken back and forth to wash the cells with PBS buffer. The PBS buffer was then slowly aspirated, and fresh culture medium was added to the washed cells. The cells were returned to a 37°C, 5% CO2 incubator for overnight culture. GFP fluorescence expression in the cells was then observed daily under an inverted fluorescence microscope. The transfected hUC-MSCs were screened using puromycin, and finally, GFP / Luc stably transfected hUC-MSCs (referred to as T-hUC-MSCs) were selected. Transfection efficiency was detected by flow cytometry; only cells with satisfactory transfection efficiency were used for subsequent experiments.

[0065] The aforementioned lentivirus solution was purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0066] The human umbilical cord mesenchymal stem cells (hUC-MSCs) cryopreserved from liquid nitrogen tanks mentioned above are of passage 5-6.

[0067] The transfection efficiency was detected by cell flow cytometry as described above, and only those with a transfection efficiency exceeding 90% can be used for subsequent experiments.

[0068] Figure 1 The transfection efficiency of the transfected human umbilical cord mesenchymal stem cells (T-hUC-MSCs) was 96.2%.

[0069] Example 2 - Cryopreservation and thawing of transfected human umbilical cord mesenchymal stem cells (T-hUC-MSCs) Cryopreservation of transfected human umbilical cord mesenchymal stem cells (T-hUC-MSCs): After the T-hUC-MSCs were prepared, they were filled into cryopreservation tubes at a rate of 0.5 mL / tube, then cooled using a programmed temperature-controlled freezer and transferred to a liquid nitrogen tank for cryopreservation.

[0070] Resuscitation and treatment: The frozen T-hUC-MSCs (CT-hUC-MSCs) were stored in liquid nitrogen for 30 days and then taken out and resuscitated in a 39 ℃ water bath.

[0071] The revived T-hUC-MSCs were transferred to a clean bench and divided into three portions. One portion of the revived T-hUC-MSCs was centrifuged to remove the cryopreservation medium, then an appropriate amount of serum-free mesenchymal stem cell culture medium was added, and the cells were transferred to a culture flask and incubated in a CO2 incubator for 24 h. Afterward, the cells were digested with trypsin and kept for later use; this portion was designated as fresh cells (FT-hUC-MSCs). Another portion of the revived T-hUC-MSCs was directly added to an appropriate amount of compound electrolyte injection solution and incubated in a CO2 incubator; this portion was designated as cryopreserved cells (C-hUC-MSCs). A third portion of the revived T-hUC-MSCs was added to an appropriate amount of mixed dilution buffer; this portion was designated as cryopreserved cells + dilution mixture (C-hUC-MSCs + CP). The T-hUC-MSCs and C-hUC-MSCs + CP were then incubated in an incubator. After incubation, cell viability was assessed using a cell counter.

[0072] All of the above operations were performed in a biosafety cabinet.

[0073] The resuscitation time of the aforementioned T-hUC-MSCs was 170 seconds.

[0074] The cell viability requirement after resuscitation is no less than 90%.

[0075] The T-hUC-MSCs were centrifuged at 400-600 rpm for 5 minutes.

[0076] The culture medium mentioned above is a serum-free culture medium for mesenchymal stem cells.

[0077] The above-mentioned trypsin concentrations are 0.5~1.0×TryPLE Select, and the EDTA concentration is 1.0 mM.

[0078] The above-mentioned diluted mixture consists of cysteine, polyvinylpyrrolidone, and compound electrolyte injection.

[0079] The concentration of the aforementioned cysteine ​​(Cys) is 5 mM.

[0080] The polyvinylpyrrolidone (PVP) mentioned above has a PVP addition amount (w / v%) of 6%.

[0081] The aforementioned compound electrolyte injection was purchased from Shanghai Baxter Medical Supplies Co., Ltd., product number: National Drug Approval Number S10940024.

[0082] The T-hUC-MSCs and C-hUC-MSCs+CP were incubated in a carbon dioxide incubator for 1 h.

[0083] The aforementioned carbon dioxide incubator was purchased from Thermo Fisher Scientific, model: HERAcell Vios160i.

[0084] Figure 2 The cell viability of human umbilical cord mesenchymal stem cells in the three groups was measured. The results showed that the cell viability of all three groups was greater than 90%, and there was no significant difference among the three groups.

[0085] Example 3 - Detection of the distribution of three groups of cells in rats using in vivo imaging of immunodeficient rats. Cell distribution assays were performed using immunodeficient rats. Fifteen SPF-grade Nude rats that passed quarantine were divided into three groups at 8-11 weeks of age: fresh cells (FT-hUC-MSCs) group (animal numbers 1101-1105), cryopreserved cells (C-hUC-MSCs) group (animal numbers 2101-2105), and cryopreserved cells + dilution mixture (C-hUC-MSCs + CP) group (animal numbers 3101-3105).

[0086] Injection of fluorescein substrate: Weigh 648 mg of fluorescein substrate into a sterile centrifuge tube, dissolve in 16 mL of physiological saline to prepare a 40 mg / mL substrate solution, and set aside. Substrate preparation should be performed in a clean bench under aseptic conditions. 10-15 minutes before the experiment, administer fluorescein sodium salt intraperitoneally at a dose of 150 mg / kg body weight. Imaging should be performed after the substrate has diffused throughout the body.

[0087] After the administration is completed, it is necessary to observe the clinical symptoms (appearance, behavior of the animals, and response to stimuli), and the death situation (time of death or time of discovery of death and pre - death responses, etc.). Observe once a day during the experiment.

[0088] At different time points, the distribution of mesenchymal stem cells in immunodeficient rats is detected by in - vivo imaging. Immunodeficient rats are anesthetized by inhaling isoflurane and placed supine on the imaging platform. In this experiment, bioluminescence imaging mode is used to image the immunodeficient rats, and the position is adjusted to ensure that the part to be detected faces the camera lens. Set parameters such as exposure time and start imaging.

[0089] Pharmacokinetic distribution experiment: After the animals are administered, observe the bioluminescence intensity and tissue distribution of the test substance in the animals on the in - vivo imager. All animals are subjected to in - vivo imaging monitoring at 0 h (before administration), 2 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, and 288 h after administration, and all animals are sacrificed after the signal approaches the background signal.

[0090] The detection method is as follows: Inject 200 mg / kg of luciferin substrate through the tail vein. The prepared concentration of the substrate is 40 mg / mL, that is, 1.35 mL is injected into each animal. Immediately after injection, induce anesthesia with 2.5% - 4.5% isoflurane and maintain anesthesia with 1.5% - 3.5% isoflurane. Use a small animal in - vivo imaging system to observe the fluorescence intensity and tissue distribution of the test substance in the animals and take pictures.

[0091] The above - mentioned immunodeficient rats are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animal production license number: SCXK (Beijing) 2021 - 0006. The experimental animal quality certificate number: No: 110011241108125348.

[0092] The above - mentioned immunodeficient rats are male rats.

[0093] The cell administration method for the above - mentioned immunodeficient rats is injection, preferably local injection, and more preferably bilateral corpus cavernosum injection. The animals are not fasted before administration and drink normal water.

[0094] The administration frequency of the above - mentioned immunodeficient rats is single - dose.

[0095] The cell concentration injected into the above - mentioned immunodeficient rats is 9×10 6 cells / mL.

[0096] The cell volume injected into the above - mentioned immunodeficient rats is 50 μL per side.

[0097] The aforementioned isoflurane was purchased from Shandong Ante Animal Husbandry Technology Co., Ltd., with batch number 2024060301.

[0098] Cell distribution results of the FT-hUC-MSCs group are shown in the figure. Figure 3 and Figure 4 .

[0099] Figure 3 and Figure 4 The experimental results showed that a single injection of Nude Rat into the corpora cavernosa of the penis yielded 9.0 × 10⁻⁶ cells. 5 After FT-hUC-MSCs were administered, the test substance was mainly distributed at the administration site. The signal peaked at 2 h post-administration, with a signal intensity of 2.15E+08 photons / sec. Subsequently, the signal gradually weakened, approaching the pre-administration background signal (1.63E+06 photons / sec) at 288 h, with a signal intensity of 1.20E+06 photons / sec. The bioluminescence intensity of the test substance in vivo ranged from 1.20E+06 to 2.15E+08 photons / sec from 2 h to 288 h post-administration. At 2 h post-administration, 1 / 5 of the animals had a test substance signal near the right inguinal lymph node, which approached the pre-administration background signal at 48 h post-administration.

[0100] Cell distribution results of the CT-hUC-MSCs group are shown in the figure. Figure 5 and Figure 6 .

[0101] Figure 5 and Figure 6 The experimental results showed that a single injection of Nude Rat into the corpora cavernosa of the penis yielded 9.0 × 10⁻⁶ cells. 5 After CT-hUC-MSCs were administered, the test substance was only distributed at the administration site. As time progressed, the bioluminescence signal at the administration site gradually increased, reaching a peak at 48 h with a signal intensity of 1.70E+08 photons / sec. Subsequently, the signal continuously weakened, approaching the pre-administration background signal (1.08E+06 photons / sec) at 288 h with a signal intensity of 9.89E+05 photons / sec. The bioluminescence intensity of the test substance in vivo ranged from 9.89E+05 to 1.70E+08 photons / sec from 2 h to 288 h after administration.

[0102] Comparing the survival of FT-hUC-MSCs and CT-hUC-MSCs in animals, it was found that the luminescence intensity of CT-hUC-MSCs in the corpus cavernosum of the penis first increased and then decreased (presumably because the luciferase protein in the cells had not yet reached its working state after the cryopreserved cells had just been thawed), with the peak time being 48 h after drug administration. In contrast, the luminescence intensity of FT-hUC-MSCs in the corpus cavernosum of the penis peaked at the first monitoring point (2 h after drug administration) and then gradually decreased. The peak signal intensities of FT-hUC-MSCs and CT-hUC-MSCs in animals were comparable. The signal intensity of both cell types decreased to near the background value 288 h after drug administration.

[0103] Cell distribution results of the CT-hUC-MSCs+CP group are shown in the figure. Figure 7 and Figure 8 .

[0104] Figure 7 and Figure 8 The experimental results showed that a single injection of 9.0 × 10⁻⁶ N·m into the corpora cavernosa of the penis with Nude Rat resulted in a significant improvement. 5 After CT-hUC-MSCs were injected with CP, the test substance was only distributed at the administration site. The signal peaked at 2 h post-administration, with a signal intensity of 1.53E+08 photons / sec. Subsequently, the signal gradually weakened, and at 288 h, it approached the pre-administration background signal (1.14E+06 photons / sec), with a signal intensity of 1.01E+06 photons / sec. The bioluminescence intensity of the test substance in vivo ranged from 1.01E+06 to 1.53E+08 photons / sec from 2 h to 288 h post-administration.

[0105] Comparing the survival of CT-hUC-MSCs and CT-hUC-MSCs+CP in animals, it was found that the luminescence intensity of CT-hUC-MSCs in the corpus cavernosum of the penis first increased and then decreased, with the peak time being 48 h after administration. In contrast, the luminescence intensity of CT-hUC-MSCs+CP in the corpus cavernosum of the penis peaked at the first monitoring point (2 h after administration) and then gradually decreased. The peak signal intensities of FT-hUC-MSCs and CT-hUC-MSCs in animals were comparable. The signal intensity of both cell types decreased to near the background value 288 h after administration.

[0106] Comparing the survival of FT-hUC-MSCs and CT-hUC-MSCs+CP in animals, it was found that the luminescence intensity of both CT-hUC-MSCs+CP and FT-hUC-MSCs in the corpus cavernosum of the penis peaked at the first monitoring point (2 h after drug administration) and then gradually decreased; the peak signal intensity of FT-hUC-MSCs and CT-hUC-MSCs in animals was comparable; the signal intensity of both cell types decreased to near the background value 288 h after drug administration.

[0107] Through this application, the cell distribution detection results of cryopreserved human umbilical cord mesenchymal stem cells after thawing are similar to those of fresh human umbilical cord mesenchymal stem cells, which facilitates more timely and accurate cell distribution detection after thawing of cryopreserved human umbilical cord mesenchymal stem cells and avoids delays.

[0108] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0109] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0110] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0111] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A diluent for cryopreserving stem cells, characterized in that, Based on the volume of the cryopreserved stem cell diluent, the cryopreserved stem cell diluent, in addition to the compound electrolyte injection solution, also includes cysteine ​​and polyvinylpyrrolidone. The concentration of cysteine ​​in the cryopreserved stem cell diluent is 1-10 mM, and the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent is 0.02-0.1 g / mL.

2. The cryopreservation stem cell diluent as described in claim 1, characterized in that, The concentration of cysteine ​​in the cryopreserved stem cell diluent is 2-8 mM, preferably 5 mM. And / or, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent is 0.03 to 0.08 g / mL, preferably, the concentration of polyvinylpyrrolidone in the cryopreserved stem cell diluent is 0.06 g / mL.

3. The cryopreservation stem cell diluent as described in claim 1, characterized in that, The stem cells are mesenchymal stem cells, preferably human umbilical cord mesenchymal stem cells; And / or, the cryopreserved stem cell diluent is suitable for detecting the activity and / or distribution of cryopreserved stem cells.

4. The application of the cryopreserved stem cell diluent as described in any one of claims 1 to 3 in the detection of stem cell activity and / or distribution.

5. A method for detecting the activity and / or distribution of cryopreserved stem cells, characterized in that, Add the cryopreserved stem cell diluent of claim 1 or 2 to the stem cell suspension, incubate, inject into animals, and perform in vivo imaging detection; the stem cells carry marker proteins.

6. The method as described in claim 5, characterized in that, The stem cell suspension is a frozen and thawed stem cell suspension. And / or, the stem cell suspension includes stem cells and cryopreservation solution.

7. The method as described in claim 5, characterized in that, The volume ratio of the stem cell suspension to the cryopreserved stem cell dilution solution is (0.5-1.5):(1.5-4.5), preferably, the volume ratio of the stem cell suspension to the cryopreserved stem cell dilution solution is 1:

3.

8. The method as described in claim 5, characterized in that, The incubation time is 0.5h to 1.5h, preferably 1h.

9. The method as described in claim 5, characterized in that, The stem cells are mesenchymal stem cells, preferably human umbilical cord mesenchymal stem cells; And / or, the marker protein includes any one or more of Luc, GFP, EBFP, EYFP, mCherry, or tdTomato; And / or, the fluorescein substrate is injected prior to the detection.

10. The method as described in claim 5, characterized in that, The animal includes at least one of rats, mice, nude mice, or rabbits; And / or, the injection includes at least one of subcutaneous injection, intradermal injection, or corpus cavernosum injection, preferably, the injection is a corpus cavernosum injection, more preferably, the injection is a bilateral corpus cavernosum injection.

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