A method for inducing adipose stem cells to differentiate into dopaminergic neurons

Abstract

This invention discloses a method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons, belonging to the field of biomedical technology. Through the isolation, passage culture, and induced transformation of adipose-derived stem cells, dopaminergic neurons are obtained. This method achieves the separation and culture of a large number of adipose-derived stem cells from autologous adipose tissue. By using two specific culture media to transform the adipose-derived stem cells into dopaminergic neurons, a safe and effective batch of stable dopaminergic neurons with a high differentiation rate can be obtained. Immunofluorescence and cell counting experiments show that the dopaminergic neuron conversion rate is 90.4±0.76, and the survival rate is 98.9±0.55. The average dopamine secretion of the transformed dopaminergic neurons, detected by a dopamine ELISA kit, is 450 pg / ml. Therefore, this invention confirms that the adipose-derived stem cell extraction and transformation steps provided by this invention are mild and stable, and the extracted cells are suitable for large-scale applications.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for inducing adipose stem cells to differentiate into dopaminergic neurons. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of Lewy bodies in the substantia nigra. Clinical manifestations include rigidity, bradykinesia, motor dysfunction, resting tremor, and sleep-behavioral disorders. In my country, the prevalence of PD in people over 65 years of age is 1.7%. With the increasing prevalence due to aging, the diagnosis and treatment of PD patients deserve significant attention. Currently, the main treatment for PD involves promoting dopamine release and alleviating motor dysfunction. While medication can alleviate symptoms to some extent, long-term use leads to drug tolerance and gradual loss of efficacy. Furthermore, as the disease progresses, the dosage needs to be increased, resulting in severe side effects. Therefore, developing new, safe, and effective treatments for PD is a crucial and urgent issue that needs to be addressed.

[0003] Stem cell therapy holds great promise for the treatment of neurodegenerative diseases. Numerous previous animal experiments have demonstrated the therapeutic effects of stem cells and induced dopaminergic neurons in the treatment of Alzheimer's and Parkinson's diseases. In particular, Parkinson's disease results from the aging or apoptosis of dopamine-secreting neurons, such as those in the substantia nigra, leading to reduced or absent dopamine secretion in the nervous system. One clinical treatment option is drug therapy, where patients alleviate clinical symptoms by ingesting dopamine substitutes. However, this method has limited efficacy; as the disease progresses, the drug's effect weakens or becomes ineffective, and with increasing dosage, side effects significantly increase, leading to intolerance and treatment failure. Another treatment method is deep brain stimulation (DBS), commonly known as a brain pacemaker. Under the stimulation of deep electrodes, dopamine is produced by substantia nigra cells. However, DBS stimulation protocols vary due to individual differences, and patient selection for this treatment method has strict indications. The controller is complex to adjust, which can easily lead to overreaction of cell switching and side effects. Furthermore, fixed parameters cannot adapt to changes in the patient's condition, sometimes resulting in limited treatment effectiveness. In addition, the effect of DBS gradually weakens or disappears after a period of use, and effective treatment is not achieved. Currently, domestic and international research has yielded some preliminary results in treating Parkinson's disease through transplantation of fetal brain neural stem cells, embryonic neural stem cells, and iPSCs. However, ethical issues make it difficult to pass approval, induced cells obtained using gene editing technology are tumorigenic, and this method suffers from problems such as technical complexity, high cost, difficulty in controlling experiments, and inconsistent purity between batches. These issues have led to considerable controversy and limited the application of these stem cell methods, hindering further development. Summary of the Invention

[0004] This invention provides a method for inducing adipose stem cells to differentiate into dopaminergic neurons. After obtaining a large number of adipose stem cells by isolating and culturing autologous adipose tissue, the adipose stem cells are transformed into dopaminergic neurons by using two specific culture media. This method can realize the culture and transformation of adipose stem cells, stably isolate and expand adipose stem cells, and efficiently transform adipose stem cells into dopaminergic neurons that can secrete dopamine. This method can be widely used in the treatment of Alzheimer's disease.

[0005] The specific technical solution provided by this invention is as follows:

[0006] The present invention provides a method for inducing adipose stem cells to differentiate into dopaminergic neurons, comprising:

[0007] Autologous adipose tissue samples were obtained by liposuction and stored under sterile conditions for up to 6 hours for the isolation and culture of adipose stem cells.

[0008] The autologous adipose tissue sample was rinsed with sodium chloride saline solution, and rinsed extensively 4 to 6 times to fully remove fat fragments, oil, blood cells and local anesthetic contained in the sample;

[0009] After rinsing, the autologous adipose tissue sample was transferred into a new 50ml sterile centrifuge tube using a 25ml pipette. Then, an equal volume of lipase was used to digest the adipose tissue. After mixing by shaking, the sample was shaken in a constant temperature shaker at 37℃ for 25 minutes, maintaining a uniform rotation speed of 230r / min during the shaking process. The sample was also mixed by hand from top to bottom every 5 minutes during the shaking process.

[0010] After shaking, place it on a centrifuge for centrifugation. During centrifugation, maintain the centrifuge temperature at 28°C, the centrifuge speed at 250 r / min, and the centrifugation time at least 5 minutes.

[0011] After discarding the top layer of liquid, the autologous adipose tissue sample was resuspended in physiological saline and then screened using a 100μm molecular sieve. After centrifugation, the centrifuge temperature was maintained at 28℃, the centrifuge speed was 250r / min, and the centrifugation time was not less than 10 minutes.

[0012] After resuspending the autologous adipose tissue sample in physiological saline and rinsing it, it was centrifuged again at 250 rpm for 10 minutes. Then, adipose-derived stem cell culture medium was added and the cell concentration was adjusted to 1.5 x 10⁻⁶ cells / mL. 5 ~2.5x10 5 / cm 2 The cells were cultured for 48 hours to achieve a cell confluence of 85%–90%.

[0013] When the cell confluence reaches 85% to 90%, use a pipette to remove the culture medium from the culture flask, add PBS / physiological saline to wash the cells once, then add 1 to 2 ml of trypsin substitute to digest for 2 to 3 minutes. After observing the cells become rounded under a microscope, add 4 ml of culture medium to stop the digestion, and then gently blow and aspirate several times to allow the cultured cells to detach from the bottom of the culture flask.

[0014] The cell suspension formed by detachment was aspirated into a 15ml centrifuge tube and centrifuged at 280r / min for 10 minutes. The supernatant was discarded and 2ml of culture medium was added and mixed by pipetting. After mixing, the mixture was evenly distributed into 3 new culture flasks.

[0015] After adding an appropriate amount of culture medium to the new culture flask, gently shake the flask until the cells are evenly distributed, and then place it in a 37°C 5% carbon dioxide incubator for culture to allow the adipose stem cells to passage.

[0016] Adipose-derived stem cells that have undergone passage to the third to fifth generation were placed in gelatin-coated 6-well culture plates for induced differentiation culture at a density of 20,000 cells / well. After one day of culture, the cells were washed with physiological saline.

[0017] After cleaning, the growth medium was replaced with an induction medium containing low-glucose DMEM, 1% FBS, 0.6% B27, 250 ng / ml SHH, 100 ng / ml FGF8, 50 ng / ml bFGF and 100 g / ml L-glutamine to continue the induction differentiation culture.

[0018] On the eighth day of induced differentiation culture, the cells were washed with physiological saline and then the growth medium was replaced with DMEM low glucose, 50 ng / ml BDNF, and 100 g / ml L-glutamine conversion medium. The cells were then incubated for 4 days to induce differentiation and obtain dopaminergic neurons.

[0019] Optionally, before rinsing the autologous adipose tissue sample with sodium chloride saline, the method further includes:

[0020] 1-2 ml of autologous adipose tissue sample was extracted for infectious disease and microbial testing. After passing the test, the autologous adipose tissue sample was rinsed with sodium chloride saline.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides a method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons. This method involves the isolation, passage culture, and induced transformation of adipose-derived stem cells into dopaminergic neurons. It achieves the extraction of a large number of adipose-derived stem cells from autologous adipose tissue. By using two specific culture media to transform these stem cells into dopaminergic neurons, a safe and effective method can obtain a stable batch of dopaminergic neurons with a high differentiation rate. Immunofluorescence and cell counting experiments show that, using this method, on day 5 of primary cell culture, the survival rate of primary neurons is approximately 82%, the dopaminergic neuron transformation rate is 90.4±0.76, and the survival rate is 98.9±0.55. The average dopamine secretion of transformed dopaminergic neurons, detected using a dopamine ELISA kit, is 450 pg / ml. Therefore, the method of this invention demonstrates a high initial survival rate of primary cells during culture, confirming that the adipose-derived stem cell extraction and transformation steps provided by this invention are mild and stable. The extracted cells are suitable for large-scale applications. Furthermore, the dopaminergic neurons obtained by this method secrete a large amount of dopamine, indicating that the transformation step is highly efficient and has minimal batch-to-batch variation. Detailed Implementation

[0023] The following is a detailed description of a method for inducing adipose stem cells to differentiate into dopaminergic neurons according to an embodiment of the present invention.

[0024] Adipose-derived stem cells (ADSCs) are pluripotent cells found in human adipose tissue. Numerous experiments have demonstrated that, under certain induction conditions, adipocytes can differentiate into osteocytes, adipocytes, chondrocytes, and nerve cells. After transplantation, they exhibit excellent replacement, repair, and therapeutic effects, without tumorigenesis or immune rejection. This project, developed by our company's R&D team, innovatively utilizes autologous ADSCs, inducing them with cytokines to become dopamine-secreting neurons, which can be provided to medical institutions to treat Parkinson's disease patients. This project avoids ethical issues and the safety risks of allogeneic cell transplantation. The purified ADSCs, after transformation, yield relatively consistent, non-gene-edited, safe, stable, and suitable-for-mass culture of dopaminergic neurons, suitable for individualized treatment of Parkinson's patients, providing a stable and feasible method for clinical treatment of Parkinson's disease.

[0025] The present invention provides a method for inducing adipose stem cells to differentiate into dopaminergic neurons, comprising:

[0026] (1) Autologous adipose tissue samples were obtained by liposuction and stored under sterile conditions for up to 6 hours for the isolation and culture of adipose stem cells.

[0027] After obtaining the autologous adipose tissue sample, it is necessary to conduct infectious disease and microbial tests on the sample. 1-2 ml of autologous adipose tissue sample can be extracted for these tests. After passing the tests, the autologous adipose tissue sample is rinsed with sodium chloride saline solution before subsequent culture and differentiation.

[0028] (2) Rinse the autologous fat tissue sample with sodium chloride saline solution, and rinse thoroughly 4 to 6 times to remove fat fragments, oil, blood cells and local anesthetic contained in the sample.

[0029] (3) Use a 25ml pipette to transfer the rinsed autologous fat tissue sample into a new 50ml sterile centrifuge tube. Then use an equal volume of lipase to digest the fat tissue. After shaking and mixing, shake in a constant temperature shaker at 37℃ for 25 minutes. During the shaking process, maintain a uniform rotation speed of 230r / min and mix by hand from top to bottom every 5 minutes.

[0030] (4) After shaking, place it on a centrifuge for centrifugation. During centrifugation, maintain the centrifuge temperature at 28°C, the centrifuge speed at 250 r / min, and the centrifugation time at least 5 minutes.

[0031] (5) After discarding the top layer liquid, the autologous fat tissue sample was resuspended in physiological saline and then screened with a 100μm molecular sieve. After that, it was centrifuged again. During the centrifugation process, the temperature of the centrifuge was kept at 28℃, the speed of the centrifuge was 250r / min, and the centrifugation time was not less than 10 minutes.

[0032] (6) After resuspending the autologous adipose tissue sample in physiological saline and rinsing it, centrifuge it again at 250 r / min for 10 minutes, then add adipose stem cell culture medium and adjust the cell concentration to 1.5 x 10⁻⁶. 5 ~2.5x10 5 / cm 2 The cells were cultured for 48 hours to achieve a cell confluence of 85%–90%.

[0033] (7) When the cell fusion reaches 85% to 90%, use a pipette to remove the culture medium from the culture flask, add PBS / physiological saline to wash the cells once, then add 1 to 2 ml of trypsin substitute to digest for 2 to 3 minutes. After observing the cells become round under a microscope, add 4 ml of culture medium to stop the digestion, and then gently blow and aspirate several times to make the cultured cells detach from the bottom of the culture flask.

[0034] (8) Aspirate the cell suspension formed by detachment into a 15ml centrifuge tube, centrifuge at 280r / min for 10 minutes, discard the previous supernatant, add 2ml of culture medium and mix by blowing and aspirating, and then evenly distribute into 3 new culture flasks.

[0035] (9) After adding an appropriate amount of culture medium to the new culture flask, gently shake the culture flask. After the cells are evenly distributed, put it into a 37°C 5% carbon dioxide incubator for culture so that the adipose stem cells can be passaged.

[0036] (10) Adipose stem cells that have undergone passage to the third to fifth generation were placed in gelatin-coated 6-well culture plates for induced differentiation culture at a density of 20,000 cells / well. After one day of culture, the cells were washed with physiological saline.

[0037] (11) After cleaning, the growth medium was replaced with an induction medium containing low sugar DMEM, 1% FBS, 0.6% B27, 250 ng / ml SHH, 100 ng / ml FGF8, 50 ng / ml bFGF and 100 g / ml L-glutamine to continue the induction differentiation culture;

[0038] This invention provides a method for inducing adipose-derived stem cells (ADSCs) to differentiate into dopaminergic neurons. Third to fifth generation ADSCs are cultured in gelatin-coated 6-well plates. On the second day of induction, the cells are washed with physiological saline and then inducing culture medium containing low-glucose DMEM, 1% FBS, 0.6% B27, 250 ng / ml SHH, 100 ng / ml FGF8, 50 ng / ml bFGF, and 100 g / ml L-glutamine. This method allows for the stable induction of ADSCs into dopaminergic neurons, maintaining a high differentiation rate and ensuring the safety of the differentiated dopaminergic neurons.

[0039] (12) After the cells were induced to differentiate and cultured for eight days, they were washed with physiological saline and the growth medium was replaced with DMEM low sugar, 50 ng / ml BDNF, 100 g / ml L-glutamine conversion medium. The cells were then incubated for 4 days and induced to differentiate to obtain dopaminergic neurons.

[0040] After differentiation induction, the conversion rate of adipose-derived stem cells was determined by immunofluorescence assay to detect tyrosine hydroxylase (TH)-positive cells (a marker of dopaminergic neurons) and Hoechst staining. The number of viable cells was calculated using trypan blue staining. Finally, the dopamine content in the cell supernatant was detected using a dopamine ELISA kit.

[0041] This invention uses autologous adipose-derived stem cells to prepare dopaminergic neurons through induced culture, avoiding the use of fetal brain tissue, embryonic tissue, and gene-edited iPSCs, thus avoiding ethical issues and tumorigenicity.

[0042] This invention provides a method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons. This method involves the isolation, passage culture, and induced transformation of adipose-derived stem cells into dopaminergic neurons. It achieves the extraction of a large number of adipose-derived stem cells from autologous adipose tissue. By using two specific culture media to transform these stem cells into dopaminergic neurons, a safe and effective method can obtain a stable batch of dopaminergic neurons with a high differentiation rate. Immunofluorescence and cell counting experiments show that, using this method, on day 5 of primary cell culture, the survival rate of primary neurons is approximately 82%, the dopaminergic neuron transformation rate is 90.4±0.76, and the survival rate is 98.9±0.55. The average dopamine secretion of transformed dopaminergic neurons, detected using a dopamine ELISA kit, is 450 pg / ml. Therefore, the method of this invention demonstrates a high initial survival rate of primary cells during culture, confirming that the adipose-derived stem cell extraction and transformation steps provided by this invention are mild and stable. The extracted cells are suitable for large-scale applications. Furthermore, the dopaminergic neurons obtained by this method secrete a large amount of dopamine, indicating that the transformation step is highly efficient and has minimal batch-to-batch variation.

[0043] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons, characterized in that, The method includes: Autologous adipose tissue samples were obtained by liposuction and stored under sterile conditions for up to 6 hours for the isolation and culture of adipose stem cells. The autologous adipose tissue sample was rinsed with sodium chloride saline solution, and rinsed extensively 4 to 6 times to fully remove fat fragments, oil, blood cells and local anesthetic contained in the sample; After rinsing, the autologous adipose tissue sample was transferred into a new 50ml sterile centrifuge tube using a 25ml pipette. Then, an equal volume of lipase was used to digest the adipose tissue. After mixing by shaking, the sample was shaken in a constant temperature shaker at 37℃ for 25 minutes, maintaining a uniform rotation speed of 230r / min during the shaking process. The sample was also mixed by hand from top to bottom every 5 minutes during the shaking process. After shaking, place it on a centrifuge for centrifugation. During centrifugation, maintain the centrifuge temperature at 28°C, the centrifuge speed at 250 r / min, and the centrifugation time at least 5 minutes. After discarding the top layer of liquid, the autologous adipose tissue sample was resuspended in physiological saline and then screened using a 100μm molecular sieve. After centrifugation, the centrifuge temperature was maintained at 28℃, the centrifuge speed was 250r / min, and the centrifugation time was not less than 10 minutes. After resuspending the autologous adipose tissue sample in physiological saline and rinsing it, it was centrifuged again at 250 rpm for 10 minutes. Then, adipose-derived stem cell culture medium was added and the cell concentration was adjusted to 1.5 x 10⁻⁶ cells / mL. 5 ~2.5x10 5 / cm 2 The cells were cultured for 48 hours to achieve a cell confluence of 85%–90%. When the cell confluence reaches 85% to 90%, use a pipette to remove the culture medium from the culture flask, add PBS / physiological saline to wash the cells once, then add 1 to 2 ml of trypsin substitute to digest for 2 to 3 minutes. After observing the cells become rounded under a microscope, add 4 ml of culture medium to stop the digestion, and then gently blow and aspirate several times to allow the cultured cells to detach from the bottom of the culture flask. The cell suspension formed by detachment was aspirated into a 15ml centrifuge tube and centrifuged at 280r / min for 10 minutes. The supernatant was discarded and 2ml of culture medium was added and mixed by pipetting. After mixing, the mixture was evenly distributed into 3 new culture flasks. After adding an appropriate amount of culture medium to the new culture flask, gently shake the flask until the cells are evenly distributed, and then place it in a 37°C 5% carbon dioxide incubator for culture to allow the adipose stem cells to passage. Adipose-derived stem cells that have undergone passage to the third to fifth generation were placed in gelatin-coated 6-well culture plates for induced differentiation culture at a density of 20,000 cells / well. After one day of culture, the cells were washed with physiological saline. After cleaning, the growth medium was replaced with an induction medium containing low-glucose DMEM, 1% FBS, 0.6% B27, 250 ng / ml SHH, 100 ng / ml FGF8, 50 ng / ml bFGF and 100 g / ml L-glutamine to continue the induction differentiation culture. On the eighth day of induced differentiation culture, the cells were washed with physiological saline and then the growth medium was replaced with DMEM low glucose, 50 ng / ml BDNF, and 100 g / ml L-glutamine conversion medium. The cells were then incubated for 4 days to induce differentiation and obtain dopaminergic neurons.

2. The method according to claim 1, characterized in that, Before rinsing the autologous adipose tissue sample with sodium chloride saline, the method further includes: 1-2 ml of autologous adipose tissue sample was extracted for infectious disease and microbial testing. After passing the test, the autologous adipose tissue sample was rinsed with sodium chloride saline.