Method for improving stem cell transplantation proliferation effect through ozone pretreatment and application

Abstract

The invention relates to a stem cell pretreatment technology in the field of biomedicine, which is characterized in that stem cells are pretreated in a specific parameter window (10-80mu g / mL, 5-60min and 36-38 DEG C) by establishing an ozone concentration-time-temperature cooperative control model, so that the anti-apoptosis ability, homing efficiency and paracrine function of the stem cells are remarkably enhanced. The method breaks through the technical bottleneck of low survival rate of traditional stem cell transplantation, and avoids the carcinogenic risk of gene modification. The application covers the three fields of myocardial repair, liver regeneration and nerve injury, and the special culture medium and the kit developed in a matched manner realize standardized operation. The survival rate of the pretreated stem cells after transplantation is increased by more than two times, the VEGF secretion amount is increased by 150%, and a safe and efficient new treatment strategy is provided for clinic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and application of ozone pretreatment to enhance the proliferation effect of stem cell transplantation. Background Technology

[0002] Stem cell transplantation faces two major technical bottlenecks: first, post-transplantation microenvironmental stress leads to an apoptosis rate exceeding 60% within 72 hours; second, the proportion of cells homing to target tissues is less than 10%. While existing technologies using gene modification (such as overexpression of HIF-1α) or growth factor pre-induction can partially improve survival rates, they suffer from problems such as complex carcinogenic risk regulation and high costs.

[0003] Although ozone medicine has some applications in the field of anti-inflammation, it has the drawbacks of high concentration sensitivity and narrow window of action in regulating stem cell activity. Current technology lacks precise control over the synergistic effect of ozone exposure time and temperature, cannot balance the relationship between oxidative stress activation and cell damage, and does not involve standardized pretreatment protocols before stem cell transplantation. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the prior art, the inventors have proposed the present invention.

[0006] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for ozone pretreatment to enhance the proliferation effect of stem cell transplantation.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for enhancing the proliferation effect of stem cell transplantation through ozone pretreatment, comprising the following steps:

[0008] (a) Suspend stem cells in a basal culture medium containing 5-20% fetal bovine serum;

[0009] (b) Introduce a mixture of oxygen and trioxide (O3 / O2 = 1:9 v / v) into the closed culture system and adjust the concentration of oxygen in the culture medium in real time at 30-50 μg / mL using a gas diffusion model;

[0010] (c) Maintain the system temperature at 36-38℃ for 20-40 minutes;

[0011] (d) After terminating the treatment, add 10-50 μM N-acetylcysteine ​​to neutralize the residual free radicals.

[0012] As a preferred embodiment of the method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in this invention, wherein: in step (a), the stem cells are bone marrow mesenchymal stem cells, adipose stem cells, or umbilical cord blood stem cells, and the cell density is controlled at 1×10⁻⁶. 6 / mL±10%.

[0013] As a preferred embodiment of the method and application of ozone pretreatment to enhance the proliferation effect of stem cell transplantation according to the present invention, step (b) adopts an ozone concentration feedback device, including a gas mixing chamber, an infrared concentration sensor and a PID temperature control module.

[0014] As a preferred embodiment of the method and application of ozone pretreatment to enhance the proliferation effect of stem cell transplantation as described in this invention, the treatment process is carried out in a hypoxic environment (5% O2) and 5-20 ng / mL of stem cell factor (SCF) is added.

[0015] A stem cell pretreatment culture medium comprising: DMEM / F12 basal medium, 20 μM N-acetylcysteine, 10 ng / mL SCF, 0.5 mM sodium pyruvate, and calcium peroxide microcapsules that continuously release 30-50 μg / mL ozone.

[0016] As a preferred embodiment of the stem cell pretreatment culture medium of the present invention, the calcium peroxide microcapsules have a particle size of 5-10 μm, the chitosan coating layer has a thickness of 0.2-0.5 μm, and the trioxide is slowly released at 37°C for 60 minutes.

[0017] The application of stem cells treated by the ozone pretreatment method described in this invention to enhance the proliferation effect of stem cell transplantation in the preparation of drugs for the treatment of myocardial infarction, wherein: the number of transplanted cells is 1×10 6 / kg body weight, delivered to the peri-infarct area via coronary catheter.

[0018] As described in this invention, the method and application of ozone pretreatment to enhance stem cell transplantation proliferation and the use of treated stem cells in the preparation of liver fibrosis repair agents include: after being combined with sodium alginate hydrogel, the stem cells are injected via the portal vein; the cell density of the hydrogel is 5 × 10⁻⁶. 7 / mL.

[0019] As described in this invention, the ozone pretreatment method enhances the proliferation effect of stem cell transplantation and its application in the preparation of spinal cord injury repair agents includes: micro-injection of IL-10 antagonist (10 μg / kg) into the injury site 48 hours before transplantation.

[0020] A stem cell transplantation kit includes an ozone concentration feedback device, a stem cell pretreatment culture medium, and...

[0021] A 4°C stem cell cryopreservation solution containing 5% DMSO and 40% fetal bovine serum;

[0022] The transplantation operation navigation system includes an indication selection module and a dosage calculation algorithm.

[0023] Beneficial effects of this invention: This invention achieves triple synergistic gains by establishing a tri-ozone biological window effect model: ① Activating the NRF2 / HO-1 pathway to enhance antioxidant capacity (Bcl-2 / Bax ratio increases by 3.2 times); ② Enhancing CXCR4 membrane localization to increase homing efficiency by 180%; ③ Remodeling mitochondrial function (membrane potential increases by 2.8 times), ensuring efficient proliferation after transplantation.

[0024] The technical advantages are: ① Precise and controllable parameters (concentration error < ±3μg / mL), ② High safety (addition of NAC to neutralize free radicals, apoptosis rate < 5%), ③ Cost reduction of 90%, single treatment cost < ¥200, and the accompanying reagent kit supports standardized clinical promotion. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0028] Example 1

[0029] This embodiment provides a method and application for ozone pretreatment to enhance the proliferation effect of stem cell transplantation, specifically, its application in myocardial repair using bone marrow mesenchymal stem cells (BMSCs).

[0030] This embodiment provides a method for ozone pretreatment of BMSCs and its application in the treatment of myocardial infarction. The specific technical route includes: preparation of stem cell suspension → ozone concentration gradient treatment → pre-transplantation neutralization treatment → myocardial targeted delivery.

[0031] Preferred Implementation

[0032] Stem cell preparation:

[0033] Specifically, fourth-generation BMSCs (ScienCell#7500) were used, and the cell density was adjusted to 1×10⁶ cells / years using DMEM / F12 medium containing 10% fetal bovine serum. 6 / mL;

[0034] Preferably, pretreatment with 0.1 mM sodium pyruvate for 2 hours before inoculation is used to enhance metabolic adaptability.

[0035] Ozone treatment system:

[0036] Specifically, a closed bioreactor (gas mixing chamber 101 + infrared sensor 103 + PID temperature control module 102) is used to introduce a mixed gas of O3 / O2 = 1:9 (v / v);

[0037] Furthermore, the concentration of ozone in the culture medium was controlled at 40 μg / mL ± 3% (adjusted in real time with feedback), the temperature was 37℃ ± 0.5℃, and the treatment time was 30 minutes.

[0038] Termination and transplantation:

[0039] Specifically, add 30 μM N-acetylcysteine ​​to neutralize residual free radicals, centrifuge, and resuspend in transplantation buffer;

[0040] As one implementation method, 1×10 is injected via coronary catheter. 6 Cells / kg were applied to the peri-infarct area of ​​rat myocardial infarction (positioning accuracy ±0.5mm).

[0041] Effect verification and principle analysis

[0042] detection indicators Processing group control group 7-day transplant survival density <![CDATA[(8.5±0.7)×10 4 / cm 2 ]]> <![CDATA[(2.3±0.3)×10 4 / cm 2 ]]> Percentage of infarct area 18.7±2.1％ 35.4±3.8％ VEGF secretion 985±85pg / mL 382±45pg / mL

[0043] Mechanism of action: Western blot showed that the NRF2 nuclear translocation rate increased by 250% and the downstream HO-1 expression was upregulated by 3.1 times; confocal microscopy analysis confirmed that the mitochondrial membrane potential (JC-1 red / green ratio) increased by 2.8 times and ATP production increased by 190%; Transwell assay showed that the CXCR4 membrane localization density increased by 180% and the cell migration number reached 450±32.

[0044] Example 2

[0045] This embodiment provides a method and application for ozone pretreatment to enhance the proliferation effect of stem cell transplantation, specifically, its application in liver regeneration and fibrosis reversal using adipose-derived stem cells (ADSCs).

[0046] This embodiment is for the treatment of liver fibrosis, and its specific feature is that ADSCs are pretreated with sustained-release ozone culture medium and then transplanted in combination with sodium alginate hydrogel.

[0047] Preferred Implementation

[0048] Design of sustained-release culture medium:

[0049] Specifically, the basal culture medium was DMEM / F12, with 10 ng / mL SCF and 0.5 mM sodium pyruvate added;

[0050] Innovatively, calcium peroxide microcapsules with a particle size of 8 μm (with a chitosan coating thickness of 0.3 μm) are added to sustain trioxide release at 37°C for 60 minutes, maintaining a concentration of 50 μg / mL ± 5%.

[0051] Cell-hydrogel complex:

[0052] Preferably, the pretreated ADSCs are mixed with a 4% sodium alginate solution at a cell density of 5 × 10⁻⁶. 7 / mL;

[0053] Furthermore, a 22G catheter was used for injection via the portal vein at a flow rate of 0.5 mL / min to prevent embolism.

[0054] Joint intervention strategy:

[0055] As one implementation method, a TGF-β inhibitor (dose 10 μg / kg) was microinjected into the liver fibrosis area 24 hours before transplantation.

[0056] Effect verification and principle analysis

[0057] Time point Group ALT(U / L) α-SMA positive rate <![CDATA[Ki67 + Cell count 72h Processing group 68±9 8.2±1.3％ <![CDATA[105±11 / mm 2 <!-- 3 -->]]> control group 153±15 32.7±4.1％ <![CDATA[49±6 / mm 2 ]]>

[0058] Mechanism of action: RNA-seq showed a 62.3% inhibition rate of the TGF-β / Smad pathway and a 3.5-fold increase in the apoptosis rate of hepatic stellate cells (TUNEL staining); the microcapsule sustained-release system extended the effective action time of ozone to 2 hours (HPLC detection of residual O3 concentration).

[0059] Example 3

[0060] This embodiment provides a method and application for ozone pretreatment to enhance the proliferation effect of stem cell transplantation, specifically, its application in the repair of nerve damage caused by umbilical cord blood stem cells (UCBSCs).

[0061] This embodiment addresses the challenge of neural regeneration in the microenvironment of elderly receptors. The specific innovation lies in the combination of hypoxia-coordinated ozone pretreatment and IL-10 antagonist microenvironment modification.

[0062] Preferred Implementation

[0063] Hypoxia pretreatment system:

[0064] Specifically, ozone treatment was carried out in a 5% O2 environment (30 μg / mL, 36℃, 40 minutes);

[0065] Preferably, a pO2 sensor (accuracy ±0.1%) is integrated inside the processing chamber to dynamically adjust the gas composition.

[0066] Modification of the receptor microenvironment:

[0067] Furthermore, the damaged area was pre-injected with an IL-10 antagonist (10 μg / kg), and stem cells were transplanted 48 hours later;

[0068] As one implementation method, a stereotactic instrument is used to guide the injection of UCBSCs to the center of spinal cord injury (coordinate error ≤50μm).

[0069] Effect verification and principle analysis

[0070] Postoperative weeks BBB Sports Rating <![CDATA[NF-200 + Nerve fiber density 8 weeks 16.4±1.5 <![CDATA[125.3±8.7 / mm 2 ]]> <![CDATA[Control group: 9.8±1.0 / 51.2±4.3 / mm 2 *]]>

[0071] Mechanism of action: Single-cell sequencing reveals TUJ1 + The percentage of neural differentiation subsets reached 23.7% (compared to 8.9% in the control group), and the Notch pathway inhibition rate was 71.2%. Electrophysiological testing showed that the motor nerve conduction velocity recovered to 82.3% of the normal group (compared to only 41.6% in the control group).

[0072] This invention reveals the "dual regulatory mechanism" of low-dose ozone (10-80 μg / mL) on stem cells by establishing a biological window effect model:

[0073] Activating moderate oxidative stress: Under strictly controlled concentration-time parameters, ozone-induced reactive oxygen species (ROS) levels were maintained within the physiological stimulation range of 5-15 μM. This dose can activate the NRF2 / ARE pathway but is insufficient to induce DNA damage. This precise regulation breaks through the "all or nothing" toxicity dilemma of traditional ozone therapy and provides a new theoretical framework for stem cell pretreatment.

[0074] Remodeling Cellular Energy Metabolism: As shown in Example 1, the 2.8-fold increase in mitochondrial membrane potential is not simply due to increased energy supply, but rather to the upregulation of the electron transport chain efficiency by uncoupling protein (UCP2), decoupling ATP synthesis from ROS generation. This mechanism is further demonstrated in the liver regeneration model by a 38% reduction in glycolysis rate, ensuring the survival advantage of transplanted cells in the ischemic microenvironment.

[0075] The core contradiction facing current stem cell transplantation technology lies in the trade-off between improving survival rate and ensuring safety: although gene modification can enhance hypoxia tolerance, it carries the risk of insertion mutations that cause cancer (Reference: NatBiotechnol. 2020; 38: 215);

[0076] Growth factor pre-induction (such as VEGF treatment) is costly and has a short-lived effect (half-life < 2 hours). This invention employs a physicochemical pretreatment strategy, creatively utilizing the half-life characteristics of ozone (20-30 minutes):

[0077] Window lock-in effect: The “20-40 minute treatment time” as defined in claim 1 exactly covers the period of trioxide decay to the safety threshold (<10 μg / mL). At this time, the NAC terminator simultaneously removes residual free radicals, forming a self-limiting safety barrier.

[0078] This invention is not simply a transplantation of ozone medicine achievements, but rather a three-stage leap forward—mechanism innovation (precise regulation of oxidative stress) → technological reconstruction (parameter window model) → industrial adaptation (cryopreservation / transportation optimization)—to solve the decade-long survival rate bottleneck in the field of stem cell transplantation. Using the three major indications of cardiovascular, hepatic, and neurological diseases as a fulcrum, it demonstrates the universality and robustness of the technical solution. The claims construct a triple barrier of parameter synergy, system integration, and combined therapy, providing an unavoidable patent protection network for industrialization.

[0079] The cost of a single operation of an ozone generator is less than ¥5, which is 99% lower than that of gene editing.

[0080] The three-dimensional constraints on concentration (10–80 μg / mL), time (5–60 min), and temperature (36–38 °C) stem from the nonlinear effect verified in the examples (survival rate >90% exists only in the narrow parameter range). If this range is exceeded (e.g., concentration >100 μg / mL), the apoptosis rate increases sharply to >30%.

[0081] The kit includes a transplantation operation navigation system, whose core algorithm dynamically correlates the indication type (myocardial / hepatic / neural) with pretreatment parameters and cell dose (e.g., myocardial infarction, default 1×10⁻⁶). 6 / kg). This combination of hardware and software design forms a closed-loop technology. Simply imitating parameters outside of this system will lead to a decrease in efficacy of >50%. It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enhancing the proliferation effect of stem cell transplantation through ozone pretreatment, characterized in that: Includes the following steps: (a) Suspend stem cells in a basal culture medium containing 5-20% fetal bovine serum; (b) Introduce a mixture of oxygen and trioxide (O3 / O2 = 1:9v / v) into the closed culture system and adjust the concentration of oxygen in the culture medium in real time to 30-50 μg / mL using a gas diffusion model; (c) Maintain the system temperature at 36-38℃ and process for 20-40 minutes; (d) After terminating the treatment, add 10-50 μM N-acetylcysteine ​​to neutralize the residual free radicals.

2. The method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in claim 1, characterized in that: In step (a), the stem cells are bone marrow mesenchymal stem cells, adipose stem cells, or umbilical cord blood stem cells, and the cell density is controlled at 1×10⁻⁶. 6 / mL±10%.

3. The method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in claim 1, characterized in that: Step (b) employs an oxygen concentration feedback device, including a gas mixing chamber, an infrared concentration sensor, and a PID temperature control module.

4. The method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in claim 1, characterized in that: The treatment was carried out in a hypoxic environment (5% O2) with the addition of 5-20 ng / mL stem cell factor (SCF).

5. A stem cell pretreatment culture medium, characterized in that: It contains DMEM / F12 basal medium, 20 μM N-acetylcysteine, 10 ng / mL SCF, 0.5 mM sodium pyruvate, and calcium peroxide microcapsules that continuously release 30-50 μg / mL ozone.

6. The stem cell pretreatment culture medium as described in claim 5, characterized in that: The calcium peroxide microcapsules have a particle size of 5-10 μm and a chitosan coating thickness of 0.2-0.5 μm. They release trioxide slowly for 60 minutes at 37°C.

7. The application of stem cells treated by the method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in any one of claims 1-4 in the preparation of drugs for treating myocardial infarction, characterized in that: The number of transplanted cells was 1×10 6 / kg body weight, delivered to the peri-infarct area via coronary catheter.

8. The application of stem cells treated by the method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in any one of claims 1-4 in the preparation of liver fibrosis repair agents, characterized in that: After being combined with sodium alginate hydrogel and injected via the portal vein, the cell density of the hydrogel was 5 × 10⁻⁶. 7 / mL.

9. The application of stem cells treated by the method for enhancing stem cell transplantation proliferation through ozone pretreatment as described in any one of claims 1-4 in the preparation of spinal cord injury repair agents, characterized in that: IL-10 antagonist (10 μg / kg) was microinjected into the lesion site 48 hours before transplantation.

10. A stem cell transplantation kit, characterized in that: It includes the ozone concentration feedback device as described in claim 3, the stem cell pretreatment culture medium as described in any one of claims 5-6; and, A 4°C stem cell cryopreservation solution containing 5% DMSO and 40% fetal bovine serum; The transplantation operation navigation system includes an indication selection module and a dosage calculation algorithm.