Efficient enrichment and purification method of adipose-derived stem cell exosome

Abstract

The invention provides an efficient enrichment and purification method of adipose-derived stem cell exosomes. According to the invention, the sex determining gene is used for distinguishing and identifying the adipose-derived mesenchymal stem cells from different sources for the first time, and then the culture medium containing specific sex hormone and regulatory factors is used for realizing in-vitro amplification of the adipose-derived mesenchymal stem cells, so that convenience is provided for preparation of the adipose-derived mesenchymal stem cell exosome. According to different sources of adipose-derived stem cell exosomes, different effects of the adipose-derived stem cell exosomes are respectively verified, and the male adipose-derived stem cell exosomes can realize skin injury repair by effectively promoting fibroblast proliferation; the female adipose-derived stem cell exosome can realize nerve injury protection by regulating the nerve conduction velocity and action potential incubation period of an injured part.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and in particular to a method for the efficient enrichment and purification of exosomes from adipose stem cells. Background Technology

[0002] Adipose-derived stem cells (ADSCs) are adult mesenchymal stem cells abundant in human adipose tissue. These stem cells possess multi-lineage differentiation potential, capable of differentiating into fat, bone, cartilage, muscle, and nerve cells in vitro. Furthermore, ADSCs can secrete various bioactive substances, including proteins, peptides, and cytokines. Compared to embryonic stem cells, ADSCs, as adult stem cells, avoid related ethical issues. Compared to bone marrow stem cells, ADSCs offer several advantages. Considering these advantages, ADSCs have broad application prospects in cell therapy and regenerative medicine.

[0003] Related studies have found that adipose-derived stem cells (ADSCs) exhibit varying degrees of plasticity and differentiation potential due to differences in tissue origin, age, and donor sex. Research on the impact of donor sex on ADSC function revealed that ADSCs extracted from the muscle of female mice possess better tissue regeneration capabilities, while ADSCs from male mice showed a higher cell differentiation rate. Furthermore, ADSCs from different sexes exhibit different oxidative stress responses to inflammatory receptor tissues. These differences often lead to varying clinical efficacy of ADSCs for the same disease. The functional differences exhibited by ADSCs from different sexes suggest that they should be differentiated based on their functional differences in clinical treatment to achieve better clinical outcomes. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention provides a highly efficient method for the enrichment and purification of adipose-derived stem cell exosomes. This invention is the first to utilize sex-determining genes to differentiate and identify adipose-derived mesenchymal stem cells (ADMSCs) from different sources, and then utilizes a culture medium containing specific sex hormones and regulatory factors to achieve in vitro expansion of ADMSCs, facilitating the preparation of ADMSC exosomes. Based on ADMSC exosomes from different sources, this invention has verified their different efficacies. This invention found that male-derived ADMSC exosomes can promote skin damage repair by promoting fibroblast proliferation, while female-derived ADMSC exosomes can protect against nerve damage by regulating nerve conduction velocity and action potential latency at the injury site. These different applications further demonstrate the necessity of differentiating ADMSCs from different sources to further enhance their functional activity.

[0005] Specifically, the present invention first provides a method for identifying the origin of adipose-derived stem cells, characterized in that the method includes the following steps:

[0006] (1) Extract genomic DNA from mouse adipose mesenchymal stem cells of unknown origin;

[0007] (2) The expression levels of Kdm5d and IDO1 genes in step (1) were quantitatively detected by qPCR;

[0008] (3) Use the following formula (a) to determine whether it comes from a female or male sample.

[0009] Relative amount = Kdm5d gene expression level / IDO1 gene expression level (a)

[0010] When the relative value is ≥1, it originates from male samples; when the relative value is <1, it originates from female samples.

[0011] Preferably, the qPCR primers and probes for the Kdm5d gene are as follows:

[0012] F: 5'-GATGTTCTTCAACAGGTGGAAT-3' (SEQ ID NO. 1);

[0013] R: 5'-CAACATTCTGCAATAGTCTGCA-3' (SEQ ID NO. 2);

[0014] P:5'-AGCTCATCAACTTGAAGAGCTGTTGGAGCAGGCACAATGG-3'(SEQ ID NO.3), where 5' is labeled FAM and 3' is labeled BHQ1;

[0015] Preferably, the qPCR primers and probes for the IDO1 gene are as follows:

[0016] F:5'-TCACAATAACTTTTATTTACTG-3'(SEQ ID NO.4);

[0017] R:5'-agAATTCCTCCAGGAAATGAGA-3' (SEQ ID NO.5);

[0018] P:5'-AGTCGGAAGAGCCCTCAAATGTGGAAAGCAGAGGGACTGG-3'(SEQ ID NO.6), where 5' is labeled ROX and 3' is labeled BHQ1.

[0019] Another object of the present invention is to provide a method for in vitro expansion of adipose-derived stem cells from different sources, comprising the following steps:

[0020] (2) Take the 1×10 obtained from the separation 3 -1×105 One adipose-derived stem cell was seeded in 10-20 mL of adipose-derived mesenchymal stem cell culture medium and cultured in suspension.

[0021] (2) After 2-4 days, observe the state of adipose-derived mesenchymal stem cells in the culture dish. When the degree of fusion reaches 80-95%, perform passage treatment.

[0022] (3) The passage process includes centrifugation to collect the supernatant, washing with 0.9% physiological saline, adding 1-2 mL of adipose-derived mesenchymal stem cell culture medium to resuspend the supernatant, and seeding it in equal amounts into T-25 culture flasks for expansion culture, ready for use.

[0023] Preferably, when the adipose stem cells in step (1) are male-derived adipose stem cells, the adipose mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 10-20% FBS, 1-4mM L-glutamine, 1-2% P / S antibiotic, 1-10nM testosterone and 5-50ng / mL EGF.

[0024] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 10% FBS, 1mM L-glutamine, 1% P / S antibiotic, 1nM testosterone and 5ng / mL EGF.

[0025] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 15% FBS, 2mM L-glutamine, 1.5% P / S antibiotic, 5nM testosterone and 20ng / mL EGF.

[0026] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 20% FBS, 4mM L-glutamine, 2% P / S antibiotic, 10nM testosterone and 50ng / mL EGF.

[0027] Preferably, when the adipose stem cells in step (1) are female-derived adipose stem cells, the adipose mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 10-20% FBS, 1-4 mM L-glutamine, 1-2% P / S antibiotic, 1-10 nM 17β-estradiol and 5-50 ng / mLNGF.

[0028] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 10% FBS, 1mM L-glutamine, 1% P / S antibiotic, 1nM 17β-estradiol and 5ng / mLNGF.

[0029] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 15% FBS, 2mM L-glutamine, 1.5% P / S antibiotic, 5nM 17β-estradiol and 20ng / mLNGF.

[0030] More preferably, the adipose-derived mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which includes 20% FBS, 4mM L-glutamine, 2% P / S antibiotic, 10nM 17β-estradiol and 50ng / mLNGF.

[0031] Another object of the present invention is to provide a method for isolating exosomes from adipose-derived stem cells, comprising the following steps:

[0032] (1) Adipose-derived stem cells were expanded in vitro using the above method. After continuous culture for 48-96 h, the supernatant was collected by centrifugation at 300×g for 10-15 min at 4℃ and set aside for use.

[0033] (2) Transfer the supernatant from step (1) to a new centrifuge tube, centrifuge at 10000×g at 4℃ for 30-60 min to remove cell debris and collect the supernatant for later use;

[0034] (3) Transfer the supernatant from step (2) to a new centrifuge tube, centrifuge at 100,000×g at 4℃ for 90-120 min, remove the supernatant, and the resulting precipitate is the exosome, referred to as male-derived adipose stem cell exosome and female-derived adipose stem cell exosome, respectively.

[0035] Another object of the present invention is to provide the use of male-derived adipose stem cell exosomes in the preparation of skin damage repair products.

[0036] Preferably, the male-derived adipose stem cell exosomes achieve skin damage repair by promoting fibroblast proliferation.

[0037] Another object of the present invention is to provide the use of female-derived adipose stem cell exosomes in the preparation of protective products against nerve damage.

[0038] Preferably, the female-derived adipose-derived stem cell exosomes achieve nerve damage protection by regulating the nerve conduction velocity and action potential latency at the site of injury.

[0039] Furthermore, the product is a pharmaceutical product and also includes pharmaceutically acceptable carriers and excipients.

[0040] The advantages of this invention are as follows: This invention provides, for the first time, a method for differentiating and identifying adipose-derived mesenchymal stem cells (ADS-C) from different sources using sex-determining genes. Furthermore, it facilitates the in vitro expansion of ADS-C using a culture medium containing specific sex hormones and regulatory factors, thus providing convenience for the preparation of ADS-C exosomes. Based on the different sources of ADS-C exosomes, this invention has verified their different efficacies. This invention found that male-derived ADS-C exosomes can effectively promote fibroblast proliferation to achieve skin damage repair, while female-derived ADS-C exosomes regulate nerve conduction velocity and action potential latency at the injury site to achieve nerve damage protection. These different applications further demonstrate the necessity of differentiating ADS-C from different sources to further enhance their functional activity. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0042] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0043] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0044] Example 1

[0045] A method for identifying the origin of adipose-derived stem cells includes the following steps:

[0046] (1) Extract genomic DNA from mouse adipose mesenchymal stem cells of unknown origin;

[0047] (2) The expression levels of Kdm5d and IDO1 genes in step (1) were quantitatively detected by qPCR;

[0048] (3) Use the following formula (a) to determine whether it comes from a female or male sample.

[0049] Relative amount = Kdm5d gene expression level / IDO1 gene expression level (a)

[0050] When the relative value is ≥1, it originates from male samples; when the relative value is <1, it originates from female samples.

[0051] The qPCR primers and probes for the Kdm5d gene are as follows:

[0052] F: 5'-GATGTTCTTCAACAGGTGGAAT-3' (SEQ ID NO. 1);

[0053] R: 5'-CAACATTCTGCAATAGTCTGCA-3' (SEQ ID NO. 2);

[0054] P:5'-AGCTCATCAACTTGAAGAGCTGTTGGAGCAGGCACAATGG-3'(SEQ ID NO.3), where 5' is labeled FAM and 3' is labeled BHQ1;

[0055] The qPCR primers and probes for the IDO1 gene are as follows:

[0056] F:5'-TCACAATAACTTTTATTTACTG-3'(SEQ ID NO.4);

[0057] R:5'-agAATTCCTCCAGGAAATGAGA-3' (SEQ ID NO.5);

[0058] P:5'-AGTCGGAAGAGCCCTCAAATGTGGAAAGCAGAGGGACTGG-3'(SEQ ID NO.6), where 5' is labeled ROX and 3' is labeled BHQ1.

[0059] Example 2

[0060] Methods for in vitro expansion of adipose-derived stem cells from different sources include the following steps:

[0061] (3) Take 1×10 from the identification and separation obtained in Example 1 3 One adipose-derived stem cell was seeded in 10 mL of adipose-derived mesenchymal stem cell culture medium and cultured in suspension.

[0062] (2) After 2 days, observe the state of adipose mesenchymal stem cells in the culture dish. When the degree of fusion reaches 80%, perform passage treatment.

[0063] (3) The passage process includes centrifugation to collect the supernatant, washing with 0.9% physiological saline, adding 1 mL of adipose-derived mesenchymal stem cell culture medium to resuspend the supernatant, and seeding it in equal amounts into T-25 culture flasks for expansion culture, ready for use.

[0064] In Example 1, when the adipose-derived stem cells were identified as male, the adipose-derived mesenchymal stem cell culture medium was mainly composed of DMEM basal medium, which included 10% FBS, 2mM L-glutamine, 1% P / S antibiotic, 1nM testosterone and 5ng / mLEGF.

[0065] In Example 1, when the adipose-derived stem cells were identified as female, the adipose-derived mesenchymal stem cell culture medium was mainly composed of DMEM basal medium, which included 10% FBS, 2mM L-glutamine, 1% P / S antibiotic, 1nM 17β-estradiol and 5ng / mLNGF.

[0066] The viability of adipose-derived stem cells obtained by the above amplification method was analyzed using the conventional CCK8 method. Treatment group 1 consisted of female-derived adipose-derived stem cells obtained in vitro by the method described in this invention, treatment group 2 consisted of male-derived adipose-derived stem cells obtained in vitro by the method described in this invention, and the control group consisted of adipose-derived stem cells obtained by amplification using commercially available DMEM basal culture medium.

[0067] The results showed that, under the same culture conditions, the average number of adipose-derived stem cells in the control group was on the order of 10-1. 5 The survival rate was 75.23±3.25%, while the average number of adipose-derived stem cells in treatment groups 1 and 2 was on the order of 10. 10 The survival rates reached 90.25±2.37% and 91.54±1.36%, respectively. From both the order of magnitude and survival rate perspectives, the in vitro expansion method for adipose-derived stem cells from different sources in this invention effectively improves the in vitro expansion survival rate of adipose-derived stem cells.

[0068] Example 3

[0069] The method for isolating exosomes from adipose-derived stem cells includes the following steps:

[0070] (1) Adipose-derived stem cells were expanded in vitro using the method described in Example 2. After continuous culture for 96 hours, the supernatant was collected by centrifugation at 300×g for 10 minutes at 4℃ and set aside for use.

[0071] (2) Transfer the supernatant from step (1) to a new centrifuge tube, centrifuge at 10000×g for 30 min at 4℃ to remove cell debris and collect the supernatant for later use;

[0072] (3) Transfer the supernatant from step (2) to a new centrifuge tube, centrifuge at 100,000×g for 90 min at 4℃, remove the supernatant, and the resulting precipitate is the exosome, referred to as male-derived adipose stem cell exosome and female-derived adipose stem cell exosome, respectively.

[0073] Example 4

[0074] Effects of male-derived adipose-derived stem cell exosomes on fibroblast proliferation: The MTT assay was used to detect the effects of male-derived adipose-derived stem cell exosomes on the proliferation of rat skin fibroblasts (purchased from Shanghai Kanglang Biotechnology Co., Ltd.). The results are shown in Table 1. Specific detection procedures can be followed according to the instructions of the MTT kit (purchased from Beyotime Biotechnology Research Institute, product number C0009S). The blank group contained only DMEM complete medium, the experimental group contained 25 μg / ml of male-derived adipose-derived stem cell exosomes in DMEM complete medium, and the control group contained an equal volume of DMEM complete medium. The cell volume in both the experimental and control groups was 1×10⁶ cells / ml. 5 indivual.

[0075] Table 1. Fibroblast proliferation analysis

[0076] Group OD value Blank group 0.157±0.012 experimental group 1.658±0.214 control group 0.425±0.035

[0077] The test results are shown in Table 1: The control group could not effectively promote the proliferation of fibroblasts, and its proliferation capacity was limited. However, the experimental group using the present invention could promote the proliferation of fibroblasts with high efficiency, and its proliferation-promoting effect was significantly better than that of the control group (P<0.05), with a significant difference and statistical significance.

[0078] Example 5

[0079] The protective effect of female-derived adipose-derived stem cell exosomes against nerve damage:

[0080] A left-sided facial nerve trunk compression injury model in SD rats was constructed using the methods described in existing literature (Establishment of a rat model of facial paralysis by facial nerve clipping, *Experimental Animals and Comparative Medicine*, 2016, Vol. 36, Issue (3): 190-194.). In the experimental group, 5 μl of isotonic saline containing exosomes derived from female adipose-derived mesenchymal stem cells was injected locally into the epineurium at the injury site. The control group received an equal volume of isotonic saline injected locally into the epineurium at the injury site. The blank control group consisted of healthy SD rats. After surgery, the rats were housed separately for 28 days. Nerve conduction velocity and action potential latency at the injury site were calculated using concentric needle electrodes, and facial expression changes were observed.

[0081] Table 2 Analysis of neuroprotective capabilities

[0082]

[0083]

[0084] The test results are shown in Table 2: the control group showed limited protective effect against facial nerve injury, while the experimental group using the present invention could efficiently promote facial nerve repair and form a better protective effect (P < 0.05). Furthermore, observation of facial expressions confirmed that the eyelids of the experimental group rats could basically close normally 10 days after surgery, and by 28 days post-surgery, they had recovered to the same facial expressions as normal rats. In contrast, the eyelids of the control group rats could not close normally 10 days after surgery, and while they would slightly open by 28 days, they had not yet achieved full recovery. These results indicate that the female-derived adipose-derived stem cell exosomes of the present invention have a good protective effect against nerve injury.

[0085] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for identifying the origin of adipose-derived stem cells, characterized in that, The method includes the following steps: (1) Extract genomic DNA from mouse adipose mesenchymal stem cells of unknown origin; (2) The expression levels of Kdm5d and IDO1 genes in step (1) were quantitatively detected by qPCR; (3) Use the following formula (a) to determine whether it comes from a female or male sample; Relative amount = Kdm5d gene expression level / IDO1 gene expression level (a) When the relative value is ≥1, it originates from male samples; when the relative value is <1, it originates from female samples.

2. The method as described in claim 1, characterized in that, The qPCR primers and probes for the Kdm5d gene are as follows: F: 5'-GATGTTCTTCAACAGGTGGAAT-3' (SEQ ID NO. 1); R: 5'-CAACATTCTGCAATAGTCTGCA-3' (SEQ ID NO. 2); P:5'-AGCTCATCAACTTGAAGAGCTGTTGGAGCAGGCACAATGG-3'(SEQ ID NO.3), where 5' is labeled FAM and 3' is labeled BHQ1.

3. The method as described in claim 1, characterized in that, The qPCR primers and probes for the IDO1 gene are as follows: F:5'-TCACAATAACTTTTATTTACTG-3'(SEQ ID NO.4); R:5'-agAATTCCTCCAGGAAATGAGA-3' (SEQ ID NO.5); P:5'-AGTCGGAAGAGCCCTCAAATGTGGAAAGCAGAGGGACTGG-3'(SEQ ID NO.6), where 5' is labeled ROX and 3' is labeled BHQ1.

4. A method for in vitro expansion of adipose-derived stem cells from different sources, characterized in that, Includes the following steps: (1) 1×10 obtained by the method described in any one of claims 1-3 3 -1×10 5 Each adipose-derived stem cell was seeded in 10-20 mL of adipose-derived mesenchymal stem cell culture medium and cultured in suspension. (2) After 2-4 days, observe the state of adipose-derived mesenchymal stem cells in the culture dish. When the degree of fusion reaches 80-95%, perform passage treatment. (3) The passage process includes centrifugation to collect the supernatant, washing with physiological saline, adding 1-2 mL of adipose-derived mesenchymal stem cell culture medium to resuspend, and then seeding an equal amount into culture flasks for expansion culture for later use.

5. The method as described in claim 4, characterized in that, When the adipose stem cells in step (1) are male-derived adipose stem cells, the adipose mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which contains 10-20% FBS, 1-4mM L-glutamine, 1-2% P / S antibiotics, 1-10nM testosterone and 5-50ng / mL EGF.

6. The method as described in claim 4, characterized in that, When the adipose stem cells in step (1) are female-derived adipose stem cells, the adipose mesenchymal stem cell culture medium is mainly composed of DMEM basal medium, which contains 10-20% FBS, 1-4mM L-glutamine, 1-2% P / S antibiotic, 1-10nM 17β-estradiol and 5-50ng / mLNGF.

7. A method for isolating exosomes from adipose-derived stem cells of different origins, comprising the following steps: (1) Expand adipose-derived stem cells in vitro using the method described in any one of claims 4-6, culture them continuously for 48-96 h, and collect the supernatant by centrifugation at 300×g for 10-15 min at 4℃ for later use; (2) Transfer the supernatant from step (1) to a new centrifuge tube, centrifuge at 10000×g at 4℃ for 30-60 min to remove cell debris and collect the supernatant for later use; (3) Transfer the supernatant from step (2) to a new centrifuge tube, centrifuge at 100,000×g at 4℃ for 90-120 min, remove the supernatant, and the resulting precipitate is the exosome, referred to as male-derived adipose stem cell exosome and female-derived adipose stem cell exosome, respectively.

8. The use of male-derived adipose-derived stem cell exosomes in the preparation of skin damage repair products.

9. Use of female-derived adipose-derived stem cell exosomes in the preparation of protective products for nerve injury.

10. The use as described in any one of claims 8-9, characterized in that, The product is a pharmaceutical product, and also includes pharmaceutically acceptable carriers and excipients.

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