CN115418349A - Method for efficiently producing exosome derived from umbilical cord mesenchymal stem cells - Google Patents
Method for efficiently producing exosome derived from umbilical cord mesenchymal stem cells Download PDFInfo
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- CN115418349A CN115418349A CN202211291940.1A CN202211291940A CN115418349A CN 115418349 A CN115418349 A CN 115418349A CN 202211291940 A CN202211291940 A CN 202211291940A CN 115418349 A CN115418349 A CN 115418349A
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Abstract
The invention relates to a method for efficiently producing exosome derived from umbilical cord mesenchymal stem cells, which comprises the following steps: (1) When in vitro culture is carried out, culturing the umbilical cord mesenchymal stem cells in a hypoxic cell culture box by using a culture medium A; (2) Removing impurities when the cells are fused to 55-65%, and separating out culture supernatant; (3) Transferring the culture supernatant into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to at least 95%, removing impurities, and separating out the cell culture supernatant; (4) Pretreating the cell culture supernatant to obtain a centrifugal liquid containing exosomes; (5) And placing the centrifugal liquid containing the exosome in an exosome extraction system for purification to obtain an exosome purification liquid derived from umbilical cord mesenchymal stem cells. The invention can obtain the umbilical cord mesenchymal stem cell-derived exosome with high purity, high yield and good uniformity in a short time.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for efficiently producing exosomes derived from umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are derived from mesoderm, and theoretically, this type of cells can differentiate into mesoderm tissue cells and have strong proliferation and multipotential differentiation abilities.
The mesenchymal stem cells have wide sources in human bodies, such as umbilical cords, placenta, fat, bone marrow, dental pulp, umbilical cord blood and the like, and can be amplified in vitro on a large scale. The mesenchymal stem cells have strong immunoregulation function and can effectively promote the recovery of hematopoietic function in the organism. Meanwhile, the compound can be differentiated into various histiocytes such as Osteoblast (Osteoblast), adipocyte (Adipocyte), chondroblast (Chondrocyte), myocyte (Myocyte) and nerve cell (Neuron) under specific in-vitro and in-vivo conditions, and can promote the repair of pathological tissue organs. Has excellent application prospect in clinical application of various diseases. However, since mesenchymal stem cells are living cell products, there are some problems in clinical use, such as long-term safety of living cells in clinical application, and the appearance of cell host antibodies in some patients.
Research in recent years has found that mesenchymal stem cells secrete a variety of vesicular structures of different particle sizes, such as exosomes (particle size: 30-200 nm), microvesicles (particle size: 200-2000 nm) and apoptotic bodies (particle size: 500-2000 nm), extracellularly in addition to some of the above-mentioned functions. The exosome is a cystic vesicle which is originally discovered by Johnstone in the process of researching reticulocytes, the particle size is only 60nm, substances such as cholesterol, sphingomyelin and ceramide are enriched on the surface of the cystic vesicle, and substances with biological activity such as a plurality of different types of proteins (growth factors, cytokines, transcription factors, enzymes, and the like), lipids (cholesterol, sphingomyelin and the like), RNA substances (LincRNAs, mRNAs and microRNAs) and signal molecules are contained in the vesicle and combined with membrane proteins on the surface of the cell, so that the biological activity substances are selectively delivered to a receptor cell, information transmission among different cells is carried out, cell signal channels are regulated, and biological functions are exerted.
It has been reported in the literature that exosomes derived from umbilical cord mesenchymal stem cells can exert effects of resisting damage of free radicals, activating cell proliferation and migration, preventing apoptosis, repairing tissue damage and the like, and show therapeutic or alleviating effects in various disease models, such as myocardial ischemia reperfusion injury, liver ischemia reperfusion injury, skin wound healing, bronchopulmonary dysplasia, kidney injury, type ii diabetes, inflammatory bowel disease, peripheral nerve injury, graft-versus-host disease and the like. The exosome derived from the mesenchymal stem cells belongs to cell derivatives, has biological functions similar to those of living cells, has no cell activity, overcomes partial defects in the current cell treatment, and is expected to be greatly used clinically.
The quality and quantity of exosomes in clinical application are the primary consideration, and the preparation methods of exosomes are more at present. Such as continuous ultracentrifugation (Sequential ultracentrifugation), gradient ultracentrifugation (Gradient ultracentrifugation), ultrafiltration (Ultrafiltration), size exclusion chromatography (Size-exclusion chromatography). These methods all have some disadvantages in preparing exosomes: 1. continuous ultracentrifugation and gradient ultracentrifugation are time-consuming and labor-consuming, require large separation equipment, and are easy to generate protein aggregation; 2. ultrafiltration can cause membrane blockage and capture, and further the purity of the exosome is affected; 3. size exclusion chromatography requires additional methods to further enrich exosomes and the equipment is relatively expensive. 4. The above methods have a problem of poor yield.
Disclosure of Invention
The invention aims to: the invention discloses a method for efficiently producing exosomes derived from umbilical cord mesenchymal stem cells, aiming at overcoming the problems in the prior art in the secretion and purification process of exosomes derived from mesenchymal stem cells. The invention creatively adopts a serum starvation method combined with a hypoxia method to culture the mesenchymal stem cells, uses proinflammatory factors to pre-treat the mesenchymal stem cells in the process, and finally adopts a full-automatic exosome extraction system EXODUS of Shenzhen Shenzhou core biotechnology company to purify the harvested exosomes, so that the obtained exosomes have high yield, high purity and good uniformity.
The technical scheme is as follows: a method for efficiently producing exosomes derived from umbilical cord mesenchymal stem cells comprises the following steps:
(1) In the in vitro culture, the umbilical cord mesenchymal stem cells are cultured in a hypoxic cell culture box by using a culture medium A, wherein:
the culture medium A contains fetal calf serum with the concentration of 5% and bFGF with the concentration of 10 ng/mL;
(2) Observing the growth state of the cells, removing impurities when the cells are fused to 55-65%, and separating out culture supernatant;
(3) Transferring the culture supernatant obtained in the step (2) into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to at least 95%, removing impurities, and separating out the cell culture supernatant, wherein:
the serum-free culture medium contains 10ng/mL of IL-6 cytokine, 50ng/mL of TNF-alpha cytokine and 20ng/mL of HGF cytokine;
(4) Pretreating the cell culture supernatant obtained in the step (3) to obtain a centrifugal liquid containing exosomes;
(5) And (5) placing the centrifugal liquid containing the exosomes obtained in the step (4) in an exosome extraction system for purification to obtain an exosome purification liquid derived from umbilical cord mesenchymal stem cells.
Further, the oxygen concentration in the hypoxic cell culture box in the step (1) is 4-6%.
Further, in the step (1), the oxygen concentration in the hypoxic cell culture box is 5%.
Further, the culture medium A in the step (1) further comprises:
6000-7000mg/L of sodium chloride;
d-glucose 4000-5000mg/L;
400-700mg/L of L-glutamine;
100-150mg/L of glycerol;
100-150mg/L of sodium dihydrogen phosphate;
100-150mg/L of sodium pyruvate;
100-150mg/L of L-isoleucine;
l-leucine 100-150mg/L;
80-100mg/L of L-threonine;
l-valine 80-100mg/L;
l-alanine hydrochloride 50-70mg/L;
50-70mg/L of L-phenylalanine;
35-55mg/L of L-serine;
35-55mg/L of 1, 3-propylene glycol;
25-40mg/L of glycine;
10-20mg/L of L-tryptophan;
10-20mg/L of phenol red;
folic acid 1-5mg/L.
Further, the serum-free medium in step (2) is UltraCULTURETM medium or PC-1TM medium.
Further, the oxygen concentration in the hypoxic cell culture box in the step (2) is 4-6%.
Further, in the step (2), the oxygen concentration in the hypoxic cell culture box is 5%.
Further, the step (4) comprises the following steps:
(41) Centrifuging the cell culture supernatant obtained in the step (3) at 4 ℃ for 10min at 500g, and removing dead cells to obtain a centrifugate;
(42) Centrifuging the centrifugate obtained in the step (41) at 4 ℃ for 20min at 2000g to remove cell debris; then centrifuging at 4 ℃ and 12000g for 30min to remove the microbubbles to obtain a centrifugate;
(43) And filtering the centrifugate obtained in the step (42) by using a filter with the pore diameter of 0.45 mu m and a filter with the pore diameter of 0.2 mu m in sequence to obtain a centrifugate containing the exosome.
Has the advantages that: the invention can obtain the umbilical cord mesenchymal stem cell-derived exosome with high purity, high yield and good uniformity in a short time.
Drawings
FIG. 1 is a schematic diagram of the identification result of the stock solution of umbilical cord mesenchymal stem cells.
Fig. 2 is a schematic view of the identification result of the umbilical cord mesenchymal stem cell-derived exosome prepared in example 1.
FIG. 3 is a schematic diagram of Western Blotting to identify the supernatant of the umbilical cord mesenchymal stem cell-derived exosome-purified solution prepared in example 1.
The specific implementation mode is as follows:
the following describes in detail specific embodiments of the present invention.
Example 1
A method for efficiently producing an umbilical cord mesenchymal stem cell-derived exosome comprises the following steps:
(1) In the in vitro culture, the umbilical cord mesenchymal stem cells are cultured in a hypoxic cell culture box by using a culture medium A, wherein:
the culture medium A contains fetal calf serum with the concentration of 5% and bFGF with the concentration of 10 ng/mL;
(2) Observing the growth state of the cells, removing impurities when the cells are fused to 60%, and separating out culture supernatant;
(3) And (3) transferring the culture supernatant obtained in the step (2) into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to 95%, removing impurities, and separating out the cell culture supernatant, wherein:
the serum-free culture medium contains 10ng/mL of IL-6 cytokine, 50ng/mL of TNF-alpha cytokine and 20ng/mL of HGF cytokine;
(4) Pretreating the cell culture supernatant obtained in the step (3) to obtain a centrifugal liquid containing exosomes;
(5) And (3) purifying the centrifugate containing the exosomes obtained in the step (4) in an exosome extraction system (ExODUS) of the Shenzhen Shuixuan biological science and technology company) to obtain an exosome purified liquid derived from umbilical cord mesenchymal stem cells.
Further, in the step (1), the oxygen concentration in the hypoxic cell culture box is 5%.
Further, the culture medium A in the step (1) further comprises:
6400mg/L sodium chloride;
d-glucose is 4500mg/L;
584mg/L of L-glutamine;
130mg/L of glycerol;
125mg/L of sodium dihydrogen phosphate;
110mg/L of sodium pyruvate;
l-isoleucine 105mg/L;
105mg/L of L-leucine;
95mg/L of L-threonine;
94mg/L of L-valine;
l-alanine hydrochloride 66mg/L;
66mg/L of L-phenylalanine;
42mg/L of L-serine;
40mg/L of 1, 3-propylene glycol;
glycine 30mg/L;
l-tryptophan is 16mg/L;
phenol red 15mg/L;
folic acid 4mg/L.
Further, the serum-free medium in the step (2) is UltraCULTURETM culture medium.
Further, in the step (2), the oxygen concentration in the low oxygen cell incubator is 5%.
Further, the step (4) comprises the following steps:
(41) Centrifuging the cell culture supernatant obtained in the step (3) at 4 ℃ for 10min at 500g, and removing dead cells to obtain a centrifugate;
(42) Centrifuging the centrifugate obtained in the step (41) at 4 ℃ at 2000g for 20min to remove cell debris; then centrifuging at 4 ℃ and 12000g for 30min to remove the microbubbles to obtain a centrifugate;
(43) And filtering the centrifugate obtained in the step (42) by using a filter with the pore diameter of 0.45 mu m and a filter with the pore diameter of 0.2 mu m in sequence to obtain a centrifugate containing the exosome.
And (3) exosome identification:
1. NTA results:
FIG. 1 is a schematic diagram of identification results of umbilical cord mesenchymal stem cell-derived exosome stock solution in step (1). Fig. 2 is a schematic diagram showing the identification result of the umbilical cord mesenchymal stem cell-derived exosome prepared in example 1, and the specific NTA results are shown in the following table:
2. western Blotting results:
the supernatant of the umbilical cord mesenchymal stem cell-derived exosome purified solution prepared in the step (5) of the embodiment was detected by using a Western Blotting method for positive exosome markers Alix, CD9, CD81 and Mac-2BP. The results of identifying the exosome-positive markers Alix, CD9, CD81, mac-2BP are shown in fig. 3.
Example 2
A method for efficiently producing an umbilical cord mesenchymal stem cell-derived exosome comprises the following steps:
(1) In the in vitro culture, the umbilical cord mesenchymal stem cells are cultured in a hypoxic cell culture box by using a culture medium A, wherein:
the culture medium A contains fetal calf serum with the concentration of 5% and bFGF with the concentration of 10 ng/mL;
(2) Observing the growth state of the cells, removing impurities when the cells are fused to 55%, and separating out culture supernatant;
(3) And (3) transferring the culture supernatant obtained in the step (2) into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to 96%, removing impurities, and separating out the cell culture supernatant, wherein:
the serum-free culture medium contains 10ng/mL of IL-6 cytokine, 50ng/mL of TNF-alpha cytokine and 20ng/mL of HGF cytokine;
(4) Pretreating the cell culture supernatant obtained in the step (3) to obtain a centrifugal liquid containing exosomes;
(5) And (5) placing the centrifugal liquid containing the exosome obtained in the step (4) in an exosome extraction system for purification to obtain an exosome purification liquid derived from umbilical cord mesenchymal stem cells.
Further, in the step (1), the oxygen concentration in the hypoxic cell culture box is 4%.
Further, the culture medium A in the step (1) further comprises:
6000mg/L of sodium chloride;
d-glucose 4000mg/L;
400mg/L of L-glutamine;
100mg/L of glycerol;
100mg/L of sodium dihydrogen phosphate;
100mg/L of sodium pyruvate;
100mg/L of L-isoleucine;
l-leucine 100mg/L;
80mg/L of L-threonine;
l-valine 80mg/L;
l-alanine hydrochloride 50mg/L;
l-phenylalanine 50mg/L;
35mg/L of L-serine;
35mg/L of 1, 3-propylene glycol;
25mg/L of glycine;
l-tryptophan is 10mg/L;
phenol red 10mg/L;
folic acid 1mg/L.
Further, the serum-free medium in the step (2) is PC-1TM medium.
Further, in the step (2), the oxygen concentration in the hypoxic cell culture box is 4%.
Further, the step (4) comprises the following steps:
(41) Centrifuging the cell culture supernatant obtained in the step (3) at 4 ℃ for 10min at 500g, and removing dead cells to obtain a centrifugate;
(42) Centrifuging the centrifugate obtained in the step (41) at 4 ℃ for 20min at 2000g to remove cell debris; then centrifuging at 4 ℃ and 12000g for 30min to remove the microbubbles to obtain a centrifugate;
(43) And filtering the centrifugate obtained in the step (42) by using a filter with the pore diameter of 0.45 μm and a filter with the pore diameter of 0.2 μm to obtain a centrifugate containing the exosome.
Example 3
A method for efficiently producing exosomes derived from umbilical cord mesenchymal stem cells comprises the following steps:
(1) In the in vitro culture, the umbilical cord mesenchymal stem cells are cultured in a hypoxic cell culture box by using a culture medium A, wherein:
the culture medium A contains fetal calf serum with the concentration of 5% and bFGF with the concentration of 10 ng/mL;
(2) Observing the growth state of the cells, removing impurities when the cells are fused to 65%, and separating out culture supernatant;
(3) And (3) transferring the culture supernatant obtained in the step (2) into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to 97%, removing impurities, and separating out the cell culture supernatant, wherein:
the serum-free culture medium contains 10ng/mL of IL-6 cytokine, 50ng/mL of TNF-alpha cytokine and 20ng/mL of HGF cytokine;
(4) Pretreating the cell culture supernatant obtained in the step (3) to obtain a centrifugal liquid containing exosomes;
(5) And (5) placing the centrifugal liquid containing the exosomes obtained in the step (4) in an exosome extraction system for purification to obtain an exosome purification liquid derived from umbilical cord mesenchymal stem cells.
Further, in the step (1), the oxygen concentration in the low oxygen cell incubator is 6%.
Further, the culture medium A in the step (1) further comprises:
7000mg/L sodium chloride;
d-glucose 5000mg/L;
l-glutamine 700mg/L;
150mg/L of glycerol;
150mg/L of sodium dihydrogen phosphate;
150mg/L of sodium pyruvate;
l-isoleucine 150mg/L;
l-leucine 150mg/L;
100mg/L of L-threonine;
l-valine of 100mg/L;
l-alanine hydrochloride 70mg/L;
l-phenylalanine 70mg/L;
55mg/L of L-serine;
55mg/L of 1, 3-propylene glycol;
40mg/L of glycine;
l-tryptophan is 20mg/L;
phenol red 20mg/L;
folic acid 5mg/L.
Further, the serum-free medium in the step (2) is UltraCULTURETM culture medium.
Further, in the step (2), the oxygen concentration in the hypoxic cell culture box is 6%.
Further, the step (4) comprises the following steps:
(41) Centrifuging the cell culture supernatant obtained in the step (3) at 4 ℃ for 10min at 500g, and removing dead cells to obtain a centrifugate;
(42) Centrifuging the centrifugate obtained in the step (41) at 4 ℃ at 2000g for 20min to remove cell debris; then centrifuging at 4 ℃ and 12000g for 30min to remove the microbubbles to obtain a centrifugate;
(43) And filtering the centrifugate obtained in the step (42) by using a filter with the pore diameter of 0.45 mu m and a filter with the pore diameter of 0.2 mu m in sequence to obtain a centrifugate containing the exosome.
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
Claims (8)
1. A method for efficiently producing an exosome derived from umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
(1) In the in vitro culture, the umbilical cord mesenchymal stem cells are cultured in a hypoxic cell culture box by using a culture medium A, wherein:
the culture medium A contains fetal calf serum with the concentration of 5% and bFGF with the concentration of 10 ng/mL;
(2) Observing the growth state of the cells, removing impurities when the cells are fused to 55-65%, and separating out culture supernatant;
(3) Transferring the culture supernatant obtained in the step (2) into a serum-free culture medium, continuously culturing in a hypoxic cell culture box until the cells are fused to at least 95%, removing impurities, and separating out the cell culture supernatant, wherein:
the serum-free culture medium contains 10ng/mL of IL-6 cytokine, 50ng/mL of TNF-alpha cytokine and 20ng/mL of HGF cytokine;
(4) Pretreating the cell culture supernatant obtained in the step (3) to obtain a centrifugal liquid containing exosomes;
(5) And (5) placing the centrifugal liquid containing the exosomes obtained in the step (4) in an exosome extraction system for purification to obtain an exosome purification liquid derived from umbilical cord mesenchymal stem cells.
2. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 1, wherein the oxygen concentration in the hypoxic cell culture chamber in the step (1) is 4-6%.
3. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 2, wherein the oxygen concentration in the hypoxic cell culture chamber in the step (1) is 5%.
4. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 1, wherein the medium A in step (1) further comprises:
6000-7000mg/L of sodium chloride;
d-glucose 4000-5000mg/L;
400-700mg/L of L-glutamine;
100-150mg/L of glycerol;
100-150mg/L of sodium dihydrogen phosphate;
100-150mg/L of sodium pyruvate;
100-150mg/L of L-isoleucine;
l-leucine 100-150mg/L;
80-100mg/L of L-threonine;
l-valine of 80-100mg/L;
l-alanine hydrochloride 50-70mg/L;
50-70mg/L of L-phenylalanine;
35-55mg/L of L-serine;
35-55mg/L of 1, 3-propylene glycol;
25-40mg/L of glycine;
10-20mg/L of L-tryptophan;
10-20mg/L of phenol red;
folic acid 1-5mg/L.
5. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 1, wherein the serum-free medium in step (2) is ultracurltetem medium or PC-1TM medium.
6. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 1, wherein the oxygen concentration in the hypoxic cell culture chamber in the step (2) is 4-6%.
7. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 6, wherein the oxygen concentration in the low oxygen cell culture chamber in the step (2) is 5%.
8. The method for efficiently producing umbilical cord mesenchymal stem cell-derived exosomes according to claim 1, wherein the step (4) comprises the steps of:
(41) Centrifuging the cell culture supernatant obtained in the step (3) at 4 ℃ for 10min at 500g, and removing dead cells to obtain a centrifugate;
(42) Centrifuging the centrifugate obtained in the step (41) at 4 ℃ at 2000g for 20min to remove cell debris; then centrifuging at 4 ℃ and 12000g for 30min to remove the microbubbles to obtain a centrifugate;
(43) And filtering the centrifugate obtained in the step (42) by using a filter with the pore diameter of 0.45 μm and a filter with the pore diameter of 0.2 μm to obtain a centrifugate containing the exosome.
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