A method for continuous production of stem cell exosomes and paracrine active macromolecules

Abstract

The present invention discloses a method for continuous production of stem cell exosomes and paracrine active macromolecules. Specifically, a hollow fiber membrane bioreactor is used to culture and enrich different products in stages, and culture fluid is harvested in stages, thereby improving the production efficiency of stem cell exosomes and paracrine active macromolecules. Meanwhile, an ultrafiltration+affinity adsorption series cycle is used to extract exosomes, so that the volume of the separation fluid can be rapidly reduced in the early stage of separation, and the concentration of exosomes is continuously increased, thereby making the adsorption of exosomes by affinity adsorption magnetic beads more and more efficient. The exosomes are adsorbed on the surface of the magnetic beads, effectively reducing the content of free exosomes in the separation fluid, effectively avoiding blockage of the ultrafiltration membrane and loss of exosomes, overcoming the shortcomings of the ultrafiltration method, namely, slower separation speed and product loss, and at the same time, overcoming the shortcomings of the affinity adsorption magnetic bead method, namely, the inability to process large-volume samples and high cost, which is conducive to promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a method for continuously producing stem cell exosomes and paracrine active macromolecules. Background Art

[0002] As a popular research direction in recent years, stem cell therapy has been proven to have great clinical application value. Among them, mesenchymal stem cells are widely used in clinical research on various diseases due to their wide sources, strong regenerative ability and excellent immunoregulatory ability. At present, it is generally believed that mesenchymal stem cells play their main role through paracrine. Stem cell paracrine factor therapy, as a cell-free treatment method, greatly reduces the risks of rejection and ectopic differentiation after allogeneic transplantation. It has achieved remarkable results in research in many fields such as trauma repair, ischemic diseases, COVID-19 treatment, medical beauty, and facial plastic surgery.

[0003] Stem cell paracrine factors used for treatment or medical aesthetics mainly include exosomes, as well as a variety of growth factors, nutritional factors and other biomacromolecules. Although stem cell paracrine factors have shown great potential in the above research fields, their complex composition, low yield and low separation and purification efficiency have greatly limited their clinical application.

[0004] Exosomes are a type of phospholipid bilayer membrane-enclosed biological structure that is actively released to the outside of cells through a series of processes such as "endocytosis-fusion-excretion". They are 30~150 nm in diameter and can be secreted by almost all types of cells. Exosomes carry important biological molecules from maternal cells, including RNA, proteins, lipids, metabolites and other molecules, and play a vital role in cell signal transduction and cell physiological and pathological activities. In recent years, it has become a research hotspot in the field of stem cell therapy and has shown significant therapeutic effects in the treatment of many diseases. Mesenchymal stem cells can secrete a variety of active macromolecules, such as VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), NGF (nerve growth factor), HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor 1), BDNF (brain-derived neurotrophic factor), GDNF (glial-derived neurotrophic factor), IL-6 (interleukin 6), IL-11 (interleukin 11), etc. Mesenchymal stem cells secrete active macromolecules that have the functions of improving the inflammatory environment, regulating the body's immune status, inhibiting cell apoptosis, promoting cell proliferation, promoting angiogenesis, promoting neural stem cell migration and differentiation, promoting axon growth and synaptic connection formation, and promoting myelin formation. Animal models of white matter damage have shown that they can inhibit the apoptosis of host cells and transplanted cells and improve the neurological function of model mice. Animal models of myocardial infarction have shown that they can promote cardiac function recovery. Skin injury experiments have shown that they can promote wound healing.

[0005] Chinese patent CN106754639A discloses a stem cell factor induction secretion culture medium. After the cells are cultured to a certain number with complete culture medium, the complete culture medium is poured out and discarded, and replaced with an induction culture medium completely composed of small molecules to induce the cells to secrete factors. After continuing to culture for a period of time, the culture medium is harvested and the macromolecules in the culture medium are separated, thereby ensuring that the separated products are all secreted from the stem cells themselves. However, this method has low production efficiency and does not separate exosomes and other types of vesicles from the products. On the other hand, when exosomes are produced, the cells are in the late growth stage and the culture medium is lacking in nutrients. The exosomes produced may have differences in composition and function from the exosomes produced during the vigorous growth period. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a continuous production method for stem cell exosomes and paracrine active macromolecules. A hollow fiber membrane bioreactor is used to culture and enrich different products in stages, and the culture fluid is harvested in stages, thereby improving the production efficiency of stem cell exosomes and paracrine active macromolecules. At the same time, ultrafiltration and affinity adsorption are used in series to cycle for exosome extraction, which effectively reduces the content of free exosomes in the separation fluid and avoids clogging of the ultrafiltration membrane and loss of exosomes.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for continuously producing stem cell exosomes and paracrine active macromolecules, comprising the following steps:

[0009] (1) Hollow fiber membrane bioreactor 3D culture: Resuspend the in vitro subcultured stem cells in complete culture medium and inoculate them into the external space of the hollow fiber membrane bioreactor. Connect the complete culture medium tank and start the circulation pump. When the glucose consumption is greater than 80%, replace the culture medium.

[0010] (2) When the medium consumption rate tends to be stable, the stem cell exosome-rich culture medium is harvested from the hollow fiber external space, and the hollow fiber external space is filled with new complete culture medium. This step is repeated until the medium consumption rate begins to decrease significantly and the cell density of the hollow fiber external space is saturated;

[0011] (3) Replace the basic culture medium without biological macromolecules for cultivation. Collect the culture medium rich in stem cell paracrine active macromolecules from the storage tank every once in a while and replace it with new basic culture medium in equal amount. Repeat this step 2 to 3 times.

[0012] (4) Extracting, separating and purifying the stem cell exosome culture medium and stem cell paracrine active macromolecule culture medium obtained in step (2) and step (3), respectively, to obtain stem cell exosomes and stem cell paracrine active macromolecules.

[0013] Furthermore, the method for obtaining stem cell exosomes in step (4) is as follows: treating the stem cell exosome culture medium at low temperature, centrifuging to remove cell debris and protein precipitate, pre-treating the supernatant with a hollow fiber microfiltration membrane to remove large particles, cell debris and macromolecular vesicles in the culture medium, and sequentially subjecting the filtrate to ultrafiltration treatment with a hollow fiber ultrafiltration membrane and adsorption treatment with affinity magnetic beads, and cyclically performing ultrafiltration and adsorption treatment to remove biological macromolecules, small molecule impurities and pigments, and eluting the stem cell exosomes adsorbed on the affinity magnetic beads with an elution solution to obtain stem cell exosomes.

[0014] Furthermore, in step (4), the method for obtaining stem cell paracrine active macromolecules is as follows: centrifuging the culture fluid rich in stem cell paracrine active macromolecules to remove cell debris, pre-treating the supernatant by filtering through a hollow fiber microfiltration membrane to remove large particles and cell debris, and circulating the filtrate through a hollow fiber ultrafiltration membrane for ultrafiltration treatment to remove small molecular impurities and pigments, thereby obtaining stem cell paracrine active macromolecules;

[0015] Furthermore, the affinity magnetic beads are streptavidin affinity magnetic beads, and are cross-linked with biotin-labeled Tim4 antibodies.

[0016] Furthermore, the in vitro subcultured cells in step (1) are subcultured for 2 to 4 generations, and the culture conditions are 37° C. and 5% CO 2 .

[0017] Furthermore, the complete culture medium is a serum-free basal culture medium for mesenchymal stem cells + 5% serum replacement; the basal culture medium is composed of DMEM / F12 + 1.5% D-glucose + 50 μmol / L L-ascorbic acid.

[0018] Furthermore, when complete medium is used for culture, the culture conditions are 37°C, 5% CO2; when basal medium is used for culture, the culture conditions are 37°C, 5% CO2, 5% O2.

[0019] Furthermore, in step (2), the volume of the complete culture medium replaced each time gradually increases according to the glucose consumption rate.

[0020] Beneficial Effects

[0021] (1) The present invention adopts a hollow fiber membrane bioreactor to culture cells in 3D. On the one hand, the cell culture space is closer to the three-dimensional microenvironment in the body, and the secreted exosomes and active macromolecules are closer to their physiological activities in the body; on the other hand, the cell culture space and the culture medium are separated by an ultrafiltration membrane, which can effectively isolate the exosomes secreted by the cells and the exosomes in the culture medium, so that there is no need to use a culture medium that removes exosomes; while the biomacromolecules in the culture medium or the biomacromolecules secreted by the cells can pass through the ultrafiltration membrane;

[0022] (2) The continuous production method of stem cell exosomes and paracrine active macromolecules of the present invention is to culture and enrich different products in stages, harvest the culture fluid in stages, and produce exosomes during the period of vigorous cell growth, effectively ensuring the normal physiological function of exosomes. In 3D culture, cells grow in stacks, and the cell expansion period is much longer than that of 2D culture. When the number of cells reaches saturation, paracrine bioactive macromolecules are produced, effectively increasing the yield of active macromolecules.

[0023] (3) The present invention adopts ultrafiltration + affinity adsorption in series circulation to extract exosomes. In the early stage of separation, the volume of the separation liquid can be rapidly reduced, and the exosome concentration is continuously increased, thereby making the adsorption of exosomes by affinity adsorption magnetic beads more and more efficient. The exosomes are adsorbed on the surface of the magnetic beads, which effectively reduces the content of free exosomes in the separation liquid, thereby greatly reducing the "squeezing effect" of the ultrafiltration process, thereby effectively avoiding the blockage of the ultrafiltration membrane and the loss of exosomes, overcoming the shortcomings of the ultrafiltration method of slower separation speed and product loss, and overcoming the shortcomings of the affinity adsorption magnetic bead method of being unable to process large-volume samples and being expensive, and achieving significant improvements in extraction speed and recovery rate; the hollow fiber ultrafiltration membrane can be reused more than ten times through a simple automated flushing process without replacement or backwashing, and the affinity magnetic beads can be reused more than five times without repeated filling. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0025] Example 1

[0026] The composition of the complete culture medium is: serum-free basal medium for mesenchymal stem cells + 5% serum replacement;

[0027] The composition of the basal medium was: DMEM / F12 + 1.5% D-glucose + 50 μmol / L L-ascorbic acid;

[0028] Hollow fiber membrane bioreactor specifications: molecular weight cut-off 100kD (95% cut-off rate), membrane surface area 1.2m 2 , the volume of the hollow fiber external space is 70 mL; the hollow fiber membrane bioreactor is pretreated with PBS and culture medium according to the instructions;

[0029] (1) 2D culture of seed cells: Take fresh umbilical cord and separate Wharton's jelly under sterile environment, suspend tissue blocks in complete culture medium and spread them in bottles. Observe cell growth under an inverted microscope. When cells grow to 80% healed, digestion and passage should be performed. 4 / cm 2 Re-plate the flask and change the medium every 3 days. When the cells grow to 80% confluent, repeat the above subculture process. The incubator conditions are 37°C, 5% CO2;

[0030] (2) Hollow fiber membrane bioreactor 3D culture: Take 5*10 P3 cells 8, resuspended with serum-free complete medium, inoculated into the outer space of the hollow fiber membrane bioreactor, the inner space of the hollow fiber is connected to the complete medium storage tank, the initial medium volume is 250mL, the circulation pump is turned on, the pump speed is set to medium speed, the device is placed in a 37°C, 5% CO2 incubator for culture, samples are taken from the medium storage tank every 12 hours, and the glucose content is measured with a glucose meter. When 80% of the glucose is consumed, the new medium is replaced. According to the glucose consumption rate, the volume of the new medium replaced each time should be gradually increased. The medium is changed every 2 days in the first 10 days, and then it is changed once a day;

[0031] (3) On the 10th day of cultivation in the hollow fiber membrane bioreactor, the consumption rate of the culture medium tended to be stable, and the culture medium rich in exosomes was harvested from the outer space of the hollow fiber every day, about 70 mL each time, and the outer space of the hollow fiber was filled with new complete culture medium. After about 40 days, the consumption rate of the culture medium began to decrease significantly. At this time, the cell density in the outer space of the hollow fiber was saturated. The culture medium was collected for the last time to obtain the culture medium rich in stem cell exosomes, and the outer space of the hollow fiber was filled with new complete culture medium;

[0032] (4) Harvesting of stem cell paracrine macromolecules: Replace the medium in the medium storage tank with a basic medium without biological macromolecules for cultivation; transfer the device to a three-gas incubator and adjust the culture parameters to 37°C, 5% CO2, and 5% O2. Collect all the old medium from the medium storage tank every 24 hours to extract the stem cell paracrine bioactive macromolecules, replace the new basic medium with the same amount, repeat the harvesting steps at 48 hours and 72 hours, and terminate the cell culture process after 72 hours to obtain a culture fluid rich in stem cell paracrine macromolecules.

[0033] Example 2

[0034] Stem cell exosomes are obtained by the following steps:

[0035] 1) Place the culture medium rich in stem cell exosomes at low temperature (4°C refrigerator for 24 hours) to allow large molecular proteins to aggregate and precipitate, centrifuge (1200g, centrifuge for 10 minutes) to remove cell debris and protein precipitates, take 150 mL of the supernatant, detect the total protein content of the supernatant by BCA method, and then dilute with PBS to a total protein content of 50 mg / L for standby use;

[0036] 2) The supernatant after PBS dilution in step (1) is placed on a hollow fiber microfiltration membrane (pore size 0.22 μm) for pretreatment to remove large particles, cell debris and macromolecular vesicles in the culture medium;

[0037] 3) Connect the hollow fiber ultrafiltration membrane (molecular weight cutoff of 100 kD) and affinity magnetic beads (commercial streptavidin affinity magnetic beads, cross-linked biotin-labeled Tim4 antibody) in series, and circulate the filtrate after the pretreatment in step (2) for ultrafiltration and adsorption treatment until the filtrate volume is 1 / 10 of the original culture volume, and make up the volume to 150 mL with buffer, and continue to circulate for ultrafiltration and adsorption treatment until the filtrate volume is 1 / 50 of the original culture volume, and make up the volume to 150 mL with buffer, and continue to circulate for ultrafiltration and adsorption treatment until the filtrate volume is 1 / 50 of the original culture volume, stop the circulation, remove the biomacromolecules, small molecule impurities and pigments, and proceed to the next step;

[0038] 4) The exosomes adsorbed on the affinity magnetic beads are eluted using an elution buffer to obtain stem cell exosomes.

[0039] Example 3

[0040] Stem cell paracrine active macromolecules are obtained by the following steps:

[0041] 1) Centrifuge the culture medium rich in stem cell paracrine active macromolecules (1200 g, 10 min) to remove cell debris and obtain the supernatant;

[0042] 2) 150 mL of the supernatant in step 1) was placed on a hollow fiber microfiltration membrane (pore size 0.1 μm) for pretreatment to remove large particles and cell debris in the culture medium;

[0043] 3) The filtrate after the pretreatment in step (2) is ultrafiltered in a hollow fiber ultrafiltration membrane (molecular weight cutoff of 5 kD), and this step is repeated until the volume of the filtrate reaches 1 / 10 of the volume of the original culture medium, the volume is supplemented with buffer to 150 mL, and the ultrafiltration cycle is continued until the volume of the filtrate reaches 1 / 50 of the volume of the original culture medium, the volume of the filtrate is supplemented with buffer to 150 mL, and the ultrafiltration cycle is continued until the volume of the filtrate reaches 1 / 10 of the volume of the original culture medium to obtain stem cell paracrine active macromolecules.

[0044] Comparative Example 1

[0045] Unlike Example 2, in step (3) of Comparative Example 1, the hollow fiber ultrafiltration membrane (throttling molecular weight of 100 kD) is not connected in series with affinity magnetic beads, and the filtrate after pretreatment in step (2) is circulated for ultrafiltration treatment until the volume of the filtrate is 1 / 10 of the volume of the original culture solution, the volume is supplemented to 150 mL with buffer, and the ultrafiltration treatment is continued to be circulated until the volume of the filtrate is 1 / 50 of the volume of the original culture solution, the volume is supplemented to 150 mL with buffer, and the ultrafiltration treatment is continued to be circulated until the volume of the filtrate is 1 / 50 of the volume of the original culture solution, and the ultrafiltration is stopped to remove biomacromolecules, small molecule impurities and pigments; the elution step is the same as that in Example 2.

[0046] Comparative Example 2

[0047] Affinity magnetic beads (commercial streptavidin affinity magnetic beads cross-linked with biotin-labeled Tim4 antibody) were connected in series behind the hollow fiber ultrafiltration membrane device in Example 3, and the pre-treated filtrate was treated according to the ultrafiltration and adsorption alternating cycle method.

[0048] Comparative Example 3

[0049] The 2D culture of seed cells is the same as step (1) of Example 1;

[0050] Use 2D culture to produce stem cell exosomes and stem cell paracrine active macromolecules, take 5*10 8 Cells (umbilical cord), resuspended in serum-free complete medium after ultracentrifugation to remove exosomes, and inoculated into multilayer cell factories at a density of 1*10 4 / cm 2 , the volume of the culture medium is 7 L, the device is placed in a 37°C, 5% CO2 incubator for culture, and the culture fluid is harvested when the cell confluence is close to 80%; according to the formula (150 / the total amount of the culture fluid rich in exosomes harvested in Example 1) × the total amount of the culture fluid rich in exosomes harvested in Comparative Example 3, a certain volume of culture fluid is taken, and the exosomes are separated and purified according to the method of Example 2 (the total protein content of the stock solution is less than 50 mg / L, and no dilution is performed);

[0051] Then, a basic culture medium without biological macromolecules was added to the multilayer cell factory, with a culture medium volume of 2L, and the device was transferred to a three-gas incubator, and the culture parameters were adjusted to 37°C, 5% CO2, and 5% O2. All old culture medium was collected from the culture medium storage tank every 24 hours to extract stem cell paracrine bioactive macromolecules, and the new basic culture medium was replaced in equal amounts. The harvesting steps were repeated at 48 hours and 72 hours, and the cell culture process was terminated after 72 hours. The volume of the culture medium rich in stem cell paracrine active macromolecules was diluted to the total amount of the culture medium rich in stem cell paracrine active macromolecules harvested in Example 1, and 150 mL was taken and processed according to Example 3 to separate stem cell paracrine active macromolecules.

[0052] Analysis and testing:

[0053] The total protein content was determined by the BCA method, the total number of particles (50-150 nm) was determined by the NanoSight nanoparticle analyzer, and the concentration of representative biomacromolecules (VEGF, GDNF, bFGF) was determined by the ELISA method.

[0054] The results of separation and purification of stem cell exosomes in Example 2, Comparative Example 1 and Comparative Example 3 are shown in Table 1 below. The extraction speed and recovery rate of Example 2 are significantly better than those of Comparative Example 1, indicating that the separation effect after hollow fiber ultrafiltration and magnetic bead affinity adsorption cross-linking is better than ultrafiltration separation; the results of separation and purification of stem cell exosomes in Example 2 and Comparative Example 3 are shown in Table 1 below. The total amount of product in Example 2 is significantly better than that in Comparative Example 1, indicating that the exosome yield produced by 3D culture is significantly better than that of 2D culture.

[0055] The results of the purification of stem cell paracrine active macromolecules in Example 3 and Comparative Examples 2~3 are shown in Table 1 below. There is no significant difference in the extraction speed and recovery rate between Example 3 and Comparative Example 2, indicating that the culture medium rich in stem cell paracrine active macromolecules produced in Example 1 does not contain exosomes, and the protein content in the culture medium rich in stem cell paracrine active macromolecules produced in Example 1 is significantly better than that in Comparative Example 3.

[0056] Table 1 Comparison of the results of separation and purification of stem cell exosomes between Example 2 and Comparative Example 1

[0057]

[0058] Table 2 Comparison of separation and purification results of active macromolecules in stem cell exosomes

[0059] .

Claims

1. A method for continuous production of stem cell exosomes and paracrine active macromolecules, characterized in that: The following steps are involved: (1) Hollow fiber membrane bioreactor 3D culture: Resuspend the in vitro subcultured stem cells in complete culture medium and inoculate them into the external space of the hollow fiber membrane bioreactor. Connect the complete culture medium tank and start the circulation pump. When the glucose consumption is greater than 80%, replace the culture medium. (2) When the medium consumption rate tends to be stable, the stem cell exosome-rich culture medium is harvested from the hollow fiber external space, and the hollow fiber external space is filled with new complete culture medium. This step is repeated until the medium consumption rate begins to decrease significantly and the cell density of the hollow fiber external space is saturated; (3) Replace the basic culture medium without biological macromolecules for cultivation, collect the culture medium rich in stem cell paracrine active macromolecules from the storage tank every once in a while, and replace the new basic culture medium with the same amount, and repeat this step 2 to 3 times; (4) extracting, separating and purifying the stem cell exosome culture fluid and stem cell paracrine active macromolecule culture fluid obtained in step (2) and step (3), respectively, to obtain stem cell exosomes and stem cell paracrine active macromolecules; The method for obtaining stem cell exosomes in step (4) is as follows: treating the stem cell exosome culture medium at low temperature, centrifuging to remove cell debris and protein precipitate, pre-treating the supernatant with a hollow fiber microfiltration membrane to remove large particles, cell debris and macromolecular vesicles in the culture medium, and sequentially subjecting the filtrate to ultrafiltration treatment with a hollow fiber ultrafiltration membrane and adsorption treatment with affinity magnetic beads, and cyclically performing ultrafiltration and adsorption treatment to remove biological macromolecules, small molecule impurities and pigments, and eluting the stem cell exosomes adsorbed on the affinity magnetic beads with an elution solution to obtain stem cell exosomes.

2. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: The method for obtaining stem cell paracrine active macromolecules in step (4) is as follows: centrifuging the culture medium rich in stem cell paracrine active macromolecules to remove cell debris, pre-treating the supernatant by filtering it through a hollow fiber microfiltration membrane to remove large particles and cell debris, and circulating the filtrate through a hollow fiber ultrafiltration membrane for ultrafiltration treatment to remove small molecular impurities and pigments, thereby obtaining stem cell paracrine active macromolecules.

3. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: The affinity magnetic beads are streptavidin affinity magnetic beads, and are cross-linked with biotin-labeled Tim4 antibodies.

4. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: The in vitro subcultured cells described in step (1) are subcultured for 2 to 4 generations, and the culture conditions are 37° C. and 5% CO 2 .

5. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: The complete culture medium is a serum-free basal culture medium for mesenchymal stem cells + 5% serum substitute; the basal culture medium is composed of DMEM / F12 + 1.5% D-glucose + 50 μmol / L L-ascorbic acid.

6. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: When complete medium is used for culture, the culture conditions are 37°C, 5% CO2; when basal medium is used for culture, the culture conditions are 37°C, 5% CO2, 5% O2.

7. The method for continuous production of stem cell exosomes and paracrine active macromolecules according to claim 1, characterized in that: In step (2), the volume of the complete culture medium replaced each time gradually increases according to the glucose consumption rate.

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