阅读说明：本技术 一种干细胞制剂及其制备方法和应用 (Stem cell preparation and preparation method and application thereof ) 是由 高建青 张添源 黄婷 于 2020-10-21 设计创作，主要内容包括：本发明公开了一种干细胞制剂及其制备方法和应用,将目的干细胞与氧化铁纳米粒共孵育刺激培养一定时间后,通过外磁场分离去除未摄取氧化铁纳米粒的目的干细胞后,制得干细胞制剂,所述的干细胞制剂具有对损伤细胞进行选择性高效线粒体转运的能力。相比于天然干细胞制剂,本发明所述的干细胞制剂具有明显提高针对损伤细胞进行线粒体移植的能力,提高效率在2倍以上。本发明还公开了所述的干细胞制剂在制备治疗与线粒体受损相关疾病的制剂中的应用,所述的干细胞制剂可以靶向受损细胞进行线粒体移植治疗,显著降低损伤细胞内过高的活性氧水平,恢复胞内ATP供能,保护和修复损伤细胞,最终实现相关疾病的有效治疗。(The invention discloses a stem cell preparation and a preparation method and application thereof. Compared with a natural stem cell preparation, the stem cell preparation provided by the invention has the advantages that the capacity of carrying out mitochondrial transplantation on damaged cells is obviously improved, and the efficiency is improved by more than 2 times. The invention also discloses application of the stem cell preparation in preparation of preparations for treating diseases related to damaged mitochondria, the stem cell preparation can target damaged cells to carry out mitochondrial transplantation treatment, remarkably reduce overhigh reactive oxygen level in the damaged cells, recover energy supply of intracellular ATP, protect and repair the damaged cells, and finally realize effective treatment of the related diseases.)

1. A method for preparing a stem cell preparation, comprising the steps of: after co-incubation stimulation culture is carried out on target stem cells and iron oxide nanoparticles for a certain time, the target stem cells which do not take in the iron oxide nanoparticles are separated and removed through an external magnetic field, and then a stem cell preparation is prepared, wherein the stem cell preparation has the capacity of carrying out selective and efficient mitochondrial transport on damaged cells.

2. The method for preparing a stem cell preparation according to claim 1, wherein the target stem cell is a pluripotent stem cell or an adult stem cell.

3. The method for preparing a stem cell preparation according to claim 1, wherein the iron oxide nanoparticles are ferroferric oxide nanoparticles or composite nanoparticles containing ferroferric oxide.

4. The method for preparing a stem cell preparation according to claim 1, wherein the size of the iron oxide nanoparticles is 1-800 nm.

5. The method for preparing a stem cell preparation according to claim 1, wherein the co-incubation stimulating culture comprises the following specific steps:

(1) performing stimulation culture on iron oxide nanoparticles with the concentration of not more than 100 mu g/mL and target stem cells in a serum-free or serum-containing condition culture medium for 1-6 h;

(2) after the stimulated culture, washing off redundant ferric oxide nanoparticles by using a buffer solution, and continuously culturing the target stem cells for more than 12 hours by replacing the buffer solution with a fresh serum-containing conditioned medium;

(3) digesting the target stem cells continuously cultured in the step (2), re-suspending the target stem cells in a neutral phosphate buffer solution, collecting to obtain a cell suspension, placing the cell suspension in an external magnetic field for 20-40 minutes, and removing the target stem cells which do not take in the iron oxide nanoparticles to obtain the stem cell preparation.

6. A stem cell preparation produced by the production method according to any one of claims 1 to 5.

7. Use of a stem cell preparation according to claim 6 for the treatment of a disease associated with damaged mitochondria.

8. The use of claim 7, wherein the disease associated with mitochondrial damage is pulmonary fibrosis.

9. The use of claim 7, wherein the disease associated with mitochondrial damage is ischemic stroke.

10. The use of claim 7, wherein the disease associated with mitochondrial damage is spinal cord injury.

Technical Field

The invention relates to the technical field of biology, in particular to a stem cell preparation, a preparation method thereof and application thereof in targeted mitochondrial transplantation.

Background

Mitochondria are the main energy supply unit in cells, and play an important role in maintaining normal physiological functions of cells. A series of studies in recent years have shown that the occurrence or development of many diseases, such as alzheimer's disease, acute lung injury, myocardial ischemia, diabetic nephropathy, and spinal cord injury, are closely related to mitochondrial damage caused by pathological stimulation and subsequent cell physiological dysfunction. When cells are damaged by stimulation of external factors, the mitochondrial function of the cells is often abnormal, so that the electron transfer efficiency of a normal respiratory chain in the cells is reduced, the generation of Reactive Oxygen Species (ROS) is increased, the generation of mitochondrial biogenesis is reduced, the mitochondrial DNA is damaged, and the mitochondrial autophagy is inhibited. Finally, the dynamic balance of the production and consumption of intracellular ROS is broken, the normal physiological function of the cells cannot be continuously maintained, and the pathological change and the apoptosis of the cells are gradually caused.

The transplantation of healthy mitochondria into the above-mentioned mitochondria-damaged cells to restore the aerobic respiration function and the electron transfer chain efficiency of these cells, reduce the ROS production of the damaged cells, improve ATP supply and calcium buffering capacity, protect and repair the damaged cells, and thus achieve disease treatment or alleviate the progress of the disease course has been gradually proven to be a feasible novel disease treatment scheme in recent years. However, how to target healthy mitochondria to damaged cells in vivo, and maintaining the activity of these healthy mitochondria during in vivo delivery and efficient mitochondrial transport to damaged cells is an important bottleneck problem faced by current mitochondrial transplantation therapeutic strategies.

At present, the main mitochondrial transplantation treatment schemes at home and abroad are: mitochondria are extracted and purified from healthy cells in vitro, and then the mitochondria are injected to the disease part through a certain technology, thereby realizing the transplantation treatment of the mitochondria. However, this treatment scheme faces many problems and limitations in practical clinical applications. Firstly, the extraction and purification processes of mitochondria themselves have certain adverse effects on the activity of mitochondria, and the survival time of free mitochondria in vitro is very short, often only hours, which makes it necessary to inject fresh mitochondria into a patient as soon as possible to ensure the curative effect, and this also greatly limits the flexibility and operability of the therapeutic strategy in clinical application. Furthermore, the method of injecting mitochondria into the disease site is not very efficient in the efficiency of transplantation. Moreover, this direct injection of mitochondria does not effectively ensure that mitochondria are transplanted only to the damaged cells at the site of disease, and may also be taken up by some other cells, causing potential safety risks.

Some recent research results show that under pathological conditions, mitochondria can spontaneously transport between different cells, thereby playing a certain role in protecting and repairing damaged cells. Moreover, this mitochondrial transport usually occurs with low probability between normal cells, and is only triggered when the cells are pathologically stimulated. This physiological mechanism provides new possibilities for selective mitochondrial transplantation to target damaged cells through mitochondrial transport between cells. On the other hand, many studies have shown that some stem cells, such as mesenchymal stem cells or neural stem cells, have the property of homing damaged tissues, and thus, the stem cells can be used as a novel cell carrier targeting delivery system to realize efficient drug delivery to damaged cells. At present, researches show that the stem cells not only have high-efficiency mitochondrial biogenesis capability, but also have the capability of unidirectionally transporting healthy mitochondria to damaged cells. The characteristics provide possibility for preparing a donor cell preparation of mitochondria by using stem cells and realizing targeted mitochondrial transplantation of damaged cells in vivo, thereby skillfully solving the main bottleneck problem of mitochondrial transplantation treatment.

However, the efficiency of mitochondrial transport of stem cells under physiological conditions is not very efficient. Studies have shown that transport efficiency is usually less than 10%. This low mitochondrial transport efficiency makes it impossible to use natural stem cells to prepare stem cell preparations for mitochondrial transplantation therapy. On the other hand, the cell physiological dysfunction caused by damaged mitochondria is a rapidly developing process, and the continuously lost mitochondrial function can be effectively compensated only by the efficient supplement of healthy mitochondria so as to save the damaged cells which are continuously apoptotic.

Therefore, the development of a stem cell preparation with high-efficiency mitochondrial transport capacity to realize high-efficiency mitochondrial transplantation of targeting damaged cells in vivo is expected to provide a feasible solution for solving a plurality of problems faced by current mitochondrial transplantation treatment. Meanwhile, a new mitochondrial transplantation-based treatment strategy and a feasible stem cell preparation are expected to be provided for the treatment of some clinical refractory diseases, and the method has very important scientific research value and clinical treatment significance.

Disclosure of Invention

The invention aims to solve the bottleneck problems of mitochondrial activity maintenance, selective transport to damaged cells, low mitochondrial transport efficiency and the like in the existing mitochondrial transplantation treatment technology, and provides a stem cell preparation, a preparation method and application thereof.

The technical scheme provided by the invention for solving the technical problems is as follows:

a preparation method of a stem cell preparation comprises the following steps: after co-incubation stimulation culture is carried out on target stem cells and iron oxide nanoparticles for a certain time, the target stem cells which do not take in the iron oxide nanoparticles are separated and removed through an external magnetic field, and then a stem cell preparation is prepared, wherein the stem cell preparation has the capacity of carrying out selective and efficient mitochondrial transport on damaged cells.

The target stem cell is a pluripotent stem cell or an adult stem cell.

The iron oxide nanoparticles are ferroferric oxide nanoparticles or composite nanoparticles containing ferroferric oxide.

In the technical scheme, no special requirements are required for the size, the shape, the surface polymer modification and the like of the iron oxide nanoparticles, but the iron oxide nanoparticles are required to be ferroferric oxide nanoparticles or composite nanoparticles containing ferroferric oxide, so that a certain amount of ferrous ions are generated in cells after the iron oxide nanoparticles are taken up and degraded by stem cells. Meanwhile, the used iron oxide nanoparticles also need to be capable of being stably dispersed in a water phase system, efficiently taken by stem cells, have good compatibility with the stem cells, have no cytotoxicity and do not influence basic stem cell functions such as stem cell differentiation and migration.

The size of the iron oxide nanoparticles is 1-800 nm.

The iron oxide nanoparticles are preferably monodisperse ferroferric oxide nanoparticles with positive charges on the surface, and the size of the iron oxide nanoparticles is preferably 5-50 nm.

The iron oxide nanoparticles are further preferably ferrimagnetic ferroferric oxide nanocubes with positive charges on the surfaces, and the size of the iron oxide nanoparticles is further preferably 10-25 nm.

The size is the diameter of the spherical iron oxide nano-particle or the side length of the square iron oxide nano-particle.

The co-incubation comprises the following specific steps:

(1) performing stimulation culture on iron oxide nanoparticles with the concentration of not more than 100 mu g/mL and target stem cells in a serum-free or serum-containing condition culture medium for 1-6 h;

(2) after the stimulated culture, washing off redundant ferric oxide nanoparticles by using a buffer solution, and continuously culturing the target stem cells for more than 12 hours by replacing the buffer solution with a fresh serum-containing conditioned medium;

(3) digesting the target stem cells continuously cultured in the step (2), and suspending the cells in a neutral Phosphate Buffer Solution (PBS), collecting to obtain a cell suspension, placing the cell suspension in an external magnetic field for 20-40 min, and removing the target stem cells which do not take in the iron oxide nanoparticles to obtain the stem cell preparation.

The co-incubation stimulation culture of the iron oxide nanoparticles and stem cells refers to adding the iron oxide nanoparticles with proper concentration into target stem cells, carrying out co-incubation culture at 37 ℃ and 5% carbon dioxide, and after a certain time, replacing a fresh condition culture medium for continuous culture to obtain the stem cell preparation with high mitochondrial transport capacity.

The concentration of the iron oxide nanoparticles is preferably 5-80 mug/mL so as to avoid causing cytotoxicity. The concentration of the iron oxide nanoparticles is more preferably 8-12 mu g/mL, and the prepared stem cell preparation has the optimal mitochondrial transport efficiency.

The ratio of the iron oxide nanoparticles to the target stem cells is 0.2-2 mu g of iron oxide nanoparticles corresponding to every ten thousand target stem cells.

The time of the stimulation culture is preferably 1-4 h, so that the iron oxide nanoparticles can be effectively absorbed by target stem cells.

The continuous culture for more than 12 hours is a continuous culture for 12-48 hours; preferably 24-48 h, so that the ingested iron oxide nanoparticles are fully degraded into ferrous ions, and further the target stem cells are stimulated to carry out efficient mitochondrial transport to the damaged cells.

The invention also provides application of the stem cell preparation in targeted therapy of diseases related to mitochondria damage.

After the stem cells and the ferric oxide nanoparticles are cultured for a certain time by co-incubation stimulation, the stem cells and the ferric oxide nanoparticles are replaced by a fresh conditioned medium for continuous culture, and the stem cell preparation with high-efficiency mitochondrial transport capacity is prepared. After the stem cell preparation is infused back into the body, the selective and efficient mitochondrial transplantation of damaged cells can be realized, and the purpose of disease treatment is achieved.

The disease related to damaged mitochondria can be pulmonary fibrosis, cerebral apoplexy or spinal cord injury, etc.

The invention also provides an application of the stem cell preparation in mitochondrial transplantation treatment, which specifically comprises the following steps: when the stem cell preparation is infused into a human body, the stem cell preparation can automatically home to a disease part and selectively carry out mitochondrial transplantation on damaged cells to treat corresponding diseases.

The routes of the stem cell preparation to be delivered into the body can be in-situ injection, systemic injection, mediated delivery, carotid artery injection, respiratory tract inhalation and the like of a disease part.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention utilizes the special biological effect of the iron oxide nanoparticles, and can prepare the stem cell preparation with the efficient mitochondrial transport capacity by simply co-incubating and stimulating the iron oxide nanoparticles and the stem cells under certain conditions. Compared with other methods such as gene recombination at present, the preparation method is simple, stable in effect and low in cost, can be used for large-scale production of stem cell preparations, and is wide in applicability and strong in popularization.

(2) The stem cell preparation with high mitochondrial transport capacity, which is prepared by the invention, can effectively ensure the activity of transplanted mitochondria and can be frozen and stored in vitro for a long time and transported for a long distance; meanwhile, the method can realize selective mitochondrial transplantation of targeted damaged cells, thereby overcoming the difficulties of stability of mitochondria, in-vitro activity maintenance, storage and transportation, selective transplantation of targeted damaged cells in vivo and the like in the current mitochondrial transplantation treatment, and being widely applied to scientific research and clinical treatment.

Drawings

Fig. 1 to 2 are micrographs of stem cell preparations prepared by superparamagnetic ferroferric oxide nanoparticles in example 1 after double staining with prussian blue and fast red, and enlarged images thereof.

FIG. 3 is a schematic diagram of the stem cell preparation prepared by the invention for selectively carrying out efficient mitochondrial transport to damaged cells.

FIG. 4 is a fluorescent photograph showing mitochondrial transport of the stem cell preparation prepared in example 1 to damaged alveolar epithelial cells in test example 1.

FIG. 5 is a statistical graph showing the mitochondrial transport efficiency of the stem cell preparation prepared in test example 2 using example 1 to damaged alveolar epithelial cells, and compared with that of a general stem cell preparation.

FIG. 6 is a statistical comparison of the mitochondrial transport efficiency of the stem cell preparation prepared in test example 2 using example 2 to healthy and damaged alveolar epithelial cells.

Fig. 7 is a statistical graph of the efficiency of mitochondrial transport of damaged neuronal cells by stem cell preparations prepared in example 4 of test example 3 using different concentrations of ferrimagnetic ferriferrous oxide nanoparticles.

FIG. 8 is a statistical graph of mitochondrial transport efficiency of the stem cell preparation prepared in example 5 of test example 4 with injured alveolar epithelial cells after different incubation times.

FIG. 9 shows the results of cell viability recovery after mitochondrial transplantation therapy of damaged alveolar epithelial cells in vitro using the stem cell preparation of the present invention of example 1.

FIG. 10 is a graph showing intracellular mitochondrial ROS levels following mitochondrial transplantation therapy of damaged alveolar epithelial cells in vitro using the stem cell preparation of the present invention of example 1.

FIG. 11 shows the recovery level of intracellular ATP after mitochondrial transplantation therapy of bleomycin-induced damaged alveolar epithelial cells in vitro using the stem cell preparation of the present invention of example 1.

FIG. 12 is a fluorescent photograph showing mitochondrial transplantation of fibrosis-damaged alveolar epithelial cells in mice after intravenous injection of the stem cell preparation of the present invention in application example 2.

Fig. 13 is a statistical chart of survival rates of mice model with pulmonary fibrosis treated by mitochondrial transplantation using the stem cell preparations of the present invention in example 3.

Fig. 14 is a graph showing the results of intervention treatment on the development of pulmonary fibrosis in mice after mitochondrial transplantation treatment of pulmonary fibrosis model mice with the stem cell preparation of the present invention in application example 3.

Detailed Description

The invention is further described with reference to specific examples, applications and figures of the specification.

Example 1: preparation of the stem cell preparation with high mitochondrial transport capacity

Taking human umbilical cord blood-derived mesenchymal stem cells (hMSCs) as an example, the stem cell preparation with high-efficiency mitochondrial transport capacity is prepared by co-incubation and stimulation culture of superparamagnetic iron oxide nanoparticles (SPION).

The method comprises the following specific steps:

1) will be 5X 10⁵The hMSCs (supplied by Zhejiang university medical college affiliated to the first Hospital, ethical lot number 2013, No. 272) were inoculated in a culture dish of 100mm in diameter and 5% CO₂Culturing at 37 deg.CAnd (5) cultivating for 24 hours.

2) The medium of 1) above was discarded, and after rinsing twice with phosphate buffered saline (PBS, pH 7.4), 10mL of fresh stem cell conditioned medium without serum was added. Then, 30 mu g/mL (1mL) of polyethyleneimine modified superparamagnetic ferroferric oxide nanoparticles (with the diameter of 10nm, prepared by Int J Pharm,552, 2018, 443) and 5% of CO are dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 1 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 24 hours.

4) Digesting and collecting the hMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 30 minutes, and removing the hMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Example 2: preparation of the stem cell preparation with high mitochondrial transport capacity

Taking hMSCs as an example, SPION co-incubation stimulation culture is adopted to prepare a stem cell preparation with high mitochondrial transport capacity.

The method comprises the following specific steps:

1) will be 5X 10⁵Inoculating hMSCs into 100 mm-diameter culture dish with 5% CO₂And culturing at 37 ℃ for 24 h.

2) The medium of 1) above was discarded, and 10mL of fresh stem cell conditioned medium without serum was added after two rinses with PBS (pH 7.4). Then, 10 mu g/mL (1mL) of polyethyleneimine modified superparamagnetic ferroferric oxide nanoparticles (with the diameter of 10nm, prepared by referring to Int J Pharm,552, 2018, 443-doped 452) and 5% of CO are dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 1 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 48 hours.

4) Digesting and collecting the hMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 30 minutes, and removing the hMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Example 3: preparation of the stem cell preparation with high mitochondrial transport capacity

Taking hMSCs as an example, the stem cell preparation with high mitochondrial transport capacity is prepared by co-incubation stimulation culture of composite nanospheres consisting of SPION.

The method comprises the following specific steps:

1) will be 5X 10⁵Inoculating hMSCs into 100 mm-diameter culture dish with 5% CO₂And culturing at 37 ℃ for 24 h.

2) The medium of 1) above was discarded, and 10mL of fresh stem cell conditioned medium without serum was added after two rinses with PBS (pH 7.4). Then, 30. mu.g/mL (1mL) of pullulan-modified superparamagnetic iron oxide nanospheres (100 nm in diameter, prepared with reference to Chinese patent ZL 201610173934), 5% CO were dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 3 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 24 hours.

4) Digesting and collecting the hMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 30 minutes, and removing the hMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Example 4: preparation of the stem cell preparation with high mitochondrial transport capacity

Taking hMSCs as an example, the stem cell preparation with high mitochondrial transport capacity is prepared by incubating and stimulating culture with ferrimagnetic ferroferric oxide nanoparticles (FION).

The method comprises the following specific steps:

1) will be 5X 10⁵Inoculating hMSCs to5% CO in a 100mm diameter Petri dish₂And culturing at 37 ℃ for 24 h.

2) The medium of 1) above was discarded, and 10mL of fresh stem cell conditioned medium without serum was added after two rinses with PBS (pH 7.4). Then, 10 mu g/mL (1mL) of polyethyleneimine modified ferrimagnetic ferroferric oxide nanoparticles (22 nm side length, prepared according to Adv Funct Mater,2019 and 1900603) and 5% of CO are dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 1 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 24 hours.

4) Digesting and collecting the hMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 10 minutes, and removing the hMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Example 5: preparation of the stem cell preparation with high mitochondrial transport capacity

For hMSCs as an example, FION co-incubation stimulation culture is adopted to prepare a stem cell preparation with high mitochondrial transport capacity.

The method comprises the following specific steps:

1) will be 5X 10⁵Inoculating hMSCs into 100 mm-diameter culture dish with 5% CO₂And culturing at 37 ℃ for 24 h.

2) The medium of 1) above was discarded, and 10mL of fresh stem cell conditioned medium without serum was added after two rinses with PBS (pH 7.4). Then, 30 mu g/mL (1mL) of polyethyleneimine modified ferrimagnetic ferroferric oxide nanoparticles (22 nm side length, prepared according to Adv Funct Mater,2019 and 1900603) and 5% of CO are dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 3 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 48 hours.

4) Digesting and collecting the hMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 10 minutes, and removing the hMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Example 6: preparation of the stem cell preparation with high mitochondrial transport capacity

Taking rat bone marrow-derived mesenchymal stem cells (rMSCs) as an example, the stem cell preparation with high mitochondrial transport capacity is prepared by SPION co-incubation stimulation culture.

The method comprises the following specific steps:

1) will be 5X 10⁵The rMSCs were inoculated in a 100mm diameter petri dish with 5% CO₂And culturing at 37 ℃ for 24 h.

2) The medium of 1) above was discarded, and 10mL of fresh stem cell conditioned medium without serum was added after two rinses with PBS (pH 7.4). Then, 30 mu g/mL (1mL) of polyethyleneimine modified superparamagnetic ferroferric oxide nanoparticles (with the diameter of 10nm, prepared by referring to Int J Pharm,552, 2018, 443) and 5% of CO are dropwise added₂Co-incubation stimulation was performed at 37 ℃ for 1 h.

3) Discarding the serum-free conditioned medium from 2) above, rinsing twice with PBS (pH 7.4), adding fresh stem cell conditioned medium containing serum, 5% CO₂The culture was continued at 37 ℃ for 24 hours.

4) Digesting and collecting the rMSCs after the iron oxide nanoparticles in the step 3) are stimulated and cultured by adopting 0.25 wt.% of pancreatin (containing 0.02 wt.% of EDTA), placing the cell suspension in an external magnetic field for 30 minutes, and removing the rMSCs which do not take up the iron oxide nanoparticles to obtain the stem cell preparation with high mitochondrial transport capacity.

Characterization experiment

(1) Morphological characterization of stem cell preparation prepared after iron oxide nanoparticle co-incubation stimulation culture

After the stem cell preparation with high mitochondrial transport capacity prepared in example 1 was fixed with 4% paraformaldehyde, prussian blue and nuclear fast red double staining were performed, and the morphology of the prepared stem cell preparation was observed under an optical microscope.

Fig. 1-2 are micrographs of stem cell preparations prepared from SPION after double staining with prussian blue and fast red nuclei and enlarged views thereof. As can be seen from the figure, the stem cell preparation of the present invention contains many iron oxide components in the cells, and these iron oxide components are distributed only in the cytoplasm. The stem cell preparation prepared by the invention is used for mitochondrial transplantation, and a schematic diagram is shown in figure 3.

Test example 1: the stem cell preparation of the present invention is transported to mitochondria of injured cells

Taking the stem cell preparation with high mitochondrial transport capacity prepared in example 1, and adopting mitochondrial staining reagent MitoRed CMXRos (excitation wavelength 579nm, emission wavelength 599nm) was labeled at 3X 10⁴The cells were seeded on mitochondria-damaged cells (1X 10)⁵One) with a diameter of 35mm in a confocal dish. Damaged cells were previously stained with mitochondrial staining reagent MitoGreen FM (excitation 490nm, emission 516nm) label. 5% CO₂Co-incubation was carried out at 37 ℃ for 4 h. Confocal laser scanning microscopy was used to observe mitochondrial transport of the stem cell preparations of the present invention to the mitochondria-damaged cells.

The results are shown in fig. 4, and the observation result of confocal laser scanning microscope shows that the stem cell preparation of the invention can realize high-efficiency mitochondrial transport to the damaged cells.

Test example 2: selective mitochondrial transport of the stem cell preparation of the invention to injured cells

Taking Bleomycin (BLM) induced damaged murine alveolar epithelial cells (TC-1) as an example, the stem cell preparations with high mitochondrial transport capacity prepared in example 1 or 2 were used to examine their capacity and efficiency for selective mitochondrial transport against damaged cells.

The prepared product of example 1 or 2 has high efficiencyStem cell preparation with mitochondrial transport capacity, using mitochondrial staining reagent MitoRed CMXRos (excitation wavelength 579nm, emission wavelength 599nm) was labeled at 3X 10⁴The cells were seeded on mitochondria-damaged cells (1X 10)⁵Individual) in 6-well plates, with normal stem cells and healthy cells with intact mitochondria as controls. Damaged cells were previously stained with mitochondrial staining reagent MitoGreen FM (excitation 490nm, emission 516nm) label. 5% CO₂Co-incubation was carried out at 37 ℃ for 4 h. Digesting and collecting the co-incubated cells, analyzing the proportion of the two mitochondria staining reagent double-positive cell populations in the total cell population by flow cytometry, and quantitatively detecting the mitochondria transport efficiency of the stem cell preparation of the invention to mitochondria damaged cells or healthy cells, and the results are shown in figures 5 and 6.

As can be seen from fig. 5, the stem cell preparation of the present invention can increase mitochondrial transport efficiency by more than 2 times compared to the natural stem cell preparation.

As can be seen in FIG. 6, the stem cell preparation of the present invention has a characteristic of being more prone to mitochondrial transport to the mitochondria-damaged cells.

Test example 3: mitochondrial transport of the inventive Stem cell preparations to injured cells prepared with different concentrations of FION

Taking the sugar oxygen deprived damaged mouse primary neuronal cells as an example, the stem cell preparations with high mitochondrial transport capacity prepared in examples 4 and 5 using different concentrations of FION were used to examine their capacity and efficiency for selective mitochondrial transport to damaged cells.

Taking the stem cell preparation with high mitochondrial transport capacity prepared in the examples 4 and 5, adopting a mitochondrial staining reagent MitoRed CMXRos (excitation wavelength 579nm, emission wavelength 599nm)) After marking, according to 3X 10⁴The cells were seeded on pre-seeded injured nerve cells (1X 10)⁵One) in a 6-well plate. Damaged nerve cells were previously stained with mitochondrial staining reagent MitoGreen FM (excitation 490nm, emission 516nm) label. 5% CO₂Co-incubation was carried out at 37 ℃ for 4 h. Digesting and collecting the co-incubated cells, analyzing the proportion of the two mitochondria staining reagent double-positive cell populations in the total cell population by flow cytometry, and quantitatively detecting the mitochondria transport efficiency of the stem cell preparation prepared by using FION with different concentrations to damaged nerve cells or healthy cells, wherein the result is shown in FIG. 7.

As can be seen from FIG. 7, the stem cell preparations prepared by using FION with different concentrations have higher mitochondrial transport efficiency than the common stem cell preparation, and optimally, the stem cell preparation with the highest mitochondrial transport efficiency can be prepared by using FION with the concentration of 10 mug/mL.

Test example 4: the stem cell preparation of the invention is directed to selective mitochondrial transport of damaged cells after different co-incubation stimulation culture durations

Taking BLM induced damaged murine TC-1 cells as an example, the stem cell preparation with high mitochondrial transport capacity prepared in example 4 and damaged alveolar epithelial cells were cultured for different periods of co-incubation stimulation, and their mitochondrial transport capacity and efficiency for damaged cells were examined.

Taking the stem cell preparation with high mitochondrial transport capacity prepared in example 4, adopting mitochondrial staining reagent MitoRed CMXRos (excitation wavelength 579nm, emission wavelength 599nm) was labeled at 3X 10⁴The cells were seeded on mitochondria-damaged cells (1X 10)⁵Individual) in 6-well plates, with normal stem cells and healthy cells with intact mitochondria as controls. Damaged cells were previously stained with mitochondrial staining reagent MitoGreen FM (excitation 490nm, emission 516nm) label. 5% CO₂And after co-incubation culture is carried out for 4-48 h at 37 ℃, the co-incubated cells are digested and collected, the proportion of the two mitochondria staining reagent double-positive cell populations in the total cell population is analyzed by flow cytometry, the mitochondrial transport efficiency of the stem cell preparation to the damaged cells at different time points is quantitatively detected, and the result is shown in fig. 8.

As can be seen from FIG. 8, the stem cell preparation of the present invention has more efficient mitochondrial transport efficiency than the normal stem cell preparation after co-incubation stimulation culture of damaged cells for different durations, and optimally, the co-incubation duration exceeds 24h to have relatively most efficient mitochondrial transport efficiency.

Application example 1: the stem cell preparation of the invention can be used for the mitochondrial transplantation treatment of damaged cells in vitro

Taking BLM-induced damaged murine TC-1 cells as an example, the stem cell preparation with high mitochondrial transport ability prepared in example 1 was used for mitochondrial transplantation therapy.

1) The TC-1 cells were arranged at 1X 10⁵The density of each cell per well was seeded in six well plates and after the cells were attached to the wall, mitochondrial damage was induced in TC-1 cells by treatment with 10 μ g/mL BLM for 24 h.

2) The stem cell preparation with high mitochondrial transport capacity prepared in example 1 was taken and processed into 3X 10⁴Density of cells per well into culture System of TC-1 cells described above, 5% CO₂Co-incubation was carried out at 37 ℃ for 24 h.

3) The survival rate of the damaged TC-1 cells treated by the BLM after the mitochondrial transplantation treatment is detected by using an Annexin V-PI apoptosis detection kit and flow cytometry.

4) Mitochondrial ROS and intracellular ATP levels of TC-1 cells are respectively detected by using a mitochondrial superoxide fluorescence probe and an ATP detection kit, the recovery of mitochondrial function of damaged TC-1 cells treated by BLM after mitochondrial transplantation treatment by using the stem cell preparation is examined, and the result is shown in fig. 9-11.

As can be seen from FIGS. 9 to 11, the stem cell preparation with high mitochondrial transport capacity can effectively improve the survival rate of TC-1 cells after damage, remarkably improve the intracellular excessive ROS level, and recover normal mitochondrial ATP energy supply. Therefore, the stem cell preparation can be used for transplantation treatment of mitochondria, effectively recovers the mitochondrial function of damaged cells, maintains the intracellular mitochondrial homeostasis and improves the survival rate of the damaged cells.

Application example 2: the stem cell preparation provided by the invention is used for carrying out mitochondrial transplantation on in-vivo targeted damaged cells

Taking BLM-induced pulmonary fibrosis model in mice as an example, the stem cell preparation with high mitochondrial transport capacity prepared in example 1 was used to achieve mitochondrial transplantation targeting to damaged alveolar epithelial cells.

1) A pulmonary fibrosis mouse model is constructed by taking 18-22 g of male C57BL/6 mice and adopting a classical intratracheal injection BLM mode according to the dose of 0.8 mg/kg.

2) Stem cell preparations having high mitochondrial transport ability were prepared from hMSCs stably expressing Green Fluorescent Protein (GFP) according to example 1, and mitochondria of the stem cell preparations were labeled with mitochondrial fluorescent probe (MitoTracker Red CMXRos), and 5 × 10 cells per mouse were used on day 7 after the model fabrication of 1) described above⁵The individual cell doses were administered by tail vein injection. 24h after administration, mice were euthanized, lung tissue was taken, and after fixed sections, alveolar epithelial cells and DAPI-labeled nuclei were labeled with anti-cytokine 7, respectively. The distribution of the stem cell preparation of the present invention in the lung and the mitochondrial transplantation into alveolar epithelial cells were observed under a confocal laser scanning microscope, and the results are shown in fig. 12.

As can be seen from FIG. 12, the stem cell preparation of the present invention can be targeted to migrate to the periphery of alveolar epithelial cells after tail vein injection, and red fluorescence representing mitochondria of stem cells can be seen in part of alveolar epithelial cells, which indicates that the stem cell preparation with high mitochondrial transport capacity prepared by the present invention can be targeted to migrate to diseased tissues through systemic injection, and can selectively carry out mitochondrial transplantation to injured cells at diseased sites.

Application example 3: the stem cell preparation provided by the invention is used for interventional treatment of pulmonary fibrosis in vivo

Taking a BLM-induced pulmonary fibrosis model of a mouse as an example, the stem cell preparation with high mitochondrial transport capacity prepared in example 1 is used to perform mitochondrial transplantation treatment on damaged epithelial cells of a lung, thereby realizing intervention treatment on pulmonary fibrosis.

1) The stem cell preparation having high mitochondrial transport ability prepared in example 1 was administered at 5X 10 per mouse⁵Each cell dose was injected into the pulmonary fibrosis mouse model constructed in application example 2 above.

2) And observing the survival condition of the pulmonary fibrosis model mice after the treatment every day, and drawing a survival curve. On day 28 after treatment, model mice were euthanized, lung tissue was taken, fixed sections were sectioned, Masson trichrome staining was performed, lung fibrosis areas were observed and calculated, and the efficacy was evaluated, with the results shown in fig. 13 to 14.

As shown in fig. 13, the stem cell preparation prepared by the present invention can significantly improve the survival rate of the pulmonary fibrosis model mouse after being used for mitochondrial transplantation therapy.

As shown in figure 14, the stem cell preparation prepared by the invention can obviously relieve the fibrotic lesion of alveolar epithelial cells after mitochondrial transplantation treatment, reduce the collagen deposition of the lung and has good potential for pulmonary fibrosis intervention treatment.

The above embodiments are described in detail to explain the technical solutions and advantages of the present invention, and it should be understood that the above embodiments are only specific examples and applications of the present invention, and are not intended to limit the present invention, and any modifications, additions, equivalents, and the like made within the scope of the principles of the present invention should be included in the scope of the present invention.