Method for obtaining melanin particles from human melanocytes

Abstract

The invention relates to a method for obtaining melanin particles from human melanocytes. Comprising the following steps: (1) culturing melanocytes to a P3 generation, inoculating the P3 generation into a culture vessel, replacing a culture medium with a DMEM culture medium containing keratinocyte exosomes after the cells are adhered to the wall, and incubating for at least 48 hours; (2) collecting the culture supernatant, centrifuging, then absorbing and discarding the supernatant, and resuspending the precipitate by using PBS (Phosphate Buffer Solution); (3) filtering by using a 20-micron filter membrane, and removing filter residues; and (4) centrifuging the filtrate, and then absorbing and discarding the supernatant. The keratinocyte exosome is used for culturing melanocytes, melanocyte proliferation can be promoted, increase of tyrosinase activity shows that melanosome differentiation is more mature, melanin synthesis is increased, and increase of melanin particle content can lay a corresponding foundation for clinical application of melanocytes in treatment of leucoderma, premature hair whitening and other related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly relates to a method for obtaining melanin granules derived from human melanocytes. Background Art

[0002] The process of melanin synthesis in melanosomes begins with their formation and maturation: the endoplasmic reticulum and Golgi apparatus in melanocytes first generate membrane-enclosed initial vesicles (stage I melanosomes), which gradually accumulate key proteins such as tyrosinase and Pmel17 inside. The latter forms a fibrous matrix to provide a scaffold for the deposition of melanin granules. Subsequently, tyrosinase is activated under the assistance of low pH environment and copper ions, catalyzing the hydroxylation of tyrosine to L-DOPA, and further oxidizing it to dopaquinone (stage II). Dopaquinone then spontaneously polymerizes to form dopachrome, and under the regulation of dopachrome tautomerase (TRP-2), generates two intermediate products - 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Under the catalysis of TRP-1 protein, DHI and DHICA oxidatively polymerize to form eumelanin. If cysteine participates in the synthesis process, dopaquinone will combine with it to form cysteinyldopa, further forming red-yellow pheomelanin (stage III). Eumelanin and / or pheomelanin finally completely fill the melanosomes in the form of dense granules (stage IV). At this time, the activity of tyrosinase disappears, the melanosomes mature, and melanin granules are formed.

[0003] Studies have shown that the main rate-limiting enzymes in melanin biosynthesis are members of the tyrosinase family. Among them, tyrosinase is a glycoprotein that binds to copper ions, and tyrosinase-related protein-2 is an iron- and zinc-binding glycoprotein. During the process of skin melanin biosynthesis, tyrosine is hydroxylated to form dopa, which is then oxidized to dopaquinone, and dihydroxyindole is converted to indolequinone. All these reaction steps require the participation of tyrosinase. When tyrosinase is lacking or its activity changes, the above reaction process significantly slows down. Changes in tyrosinase activity may cause the onset of vitiligo and have a certain impact on hair color.

[0004] Currently, autologous melanocyte transplantation has been an effective means for treating vitiligo. However, due to the limited number of primary cultured melanocytes and the limited area of a single transplantation, it cannot well meet the treatment needs of patients with extensive vitiligo. Therefore, the industry is studying whether it is possible to directly induce the maturation of melanosomes in vitro, obtain and preserve melanin granules, and further study the possibility of using melanin granules to treat pigmentary diseases. Summary of the Invention

[0005] The purpose of the present invention is to disclose a method for obtaining melanin granules derived from human melanocytes, so as to solve one or more technical problems existing in the prior art, and provide at least one beneficial choice or create conditions.

[0006] The first aspect of the present invention is to provide a method for obtaining human-derived melanin granules.

[0007] The method for obtaining melanin granules according to the first aspect of the present invention includes the following steps: (1) Cultivate melanocytes to passage P3, inoculate them into a culture vessel, and replace the culture medium with DMEM medium containing keratinocyte exosomes after the cells adhere to the wall, and incubate for at least 48 hours; (2) Collect the culture supernatant, aspirate and discard the upper clear liquid after centrifugation, and resuspend the precipitate with PBS; (3) Filter with a 20 μm filter membrane to remove the filter residue; (4) Aspirate and discard the upper clear liquid after centrifuging the filtrate.

[0008] In a further application embodiment, the method for obtaining melanin granules further includes an improved vitrification freezing step: (5) Add a vitrification cryopreservation solution diluted with a culture medium for pre-equilibration; (6) Aspirate and discard the upper clear liquid after centrifugation, and then transfer it to a cryotube for pre-cooling at 4°C; (7) Perform vitrification freezing at a rate of decreasing 25°C per minute, and store it in a liquid nitrogen environment after completion.

[0009] In a further application embodiment, the method for obtaining melanin granules further includes a thawing and cryopreservation solution removal step after vitrification freezing: (8) Take out the cryotube in a liquid nitrogen environment, thaw it in a 37°C constant temperature water bath, add 3 volumes of PBS solution for washing after thawing, centrifuge to remove the upper clear liquid, and repeat the washing at least 2 times.

[0010] In a further application embodiment, the preparation method of the keratinocyte exosome-containing includes the steps: (a) Recover the culture supernatant after culturing keratinocytes for at least 48 hours, and filter and sterilize it with a 20 μm filter membrane; (b) Retain the supernatant after centrifuging at 2000×g for 30 minutes, then retain the precipitate after centrifuging at 100000×g for 60 minutes, and resuspend it with a PBS solution; (c) Extract and collect the exosomes in the suspension.

[0011] In a further application embodiment, the culture medium used in step (a) is EpiLife® Medium serum-free medium supplemented with 1% human keratinocyte growth supplement HKGS and 0.1 mmol / L ascorbic acid.

[0012] In a further application embodiment, the concentration of keratinocyte exosomes in the DMEM medium containing keratinocyte exosomes in step (1) is 1×108 ~1×10 10 particles / mL.

[0013] In a further application embodiment, the centrifugation condition in step (2) is centrifugation at 2000×g for 10 minutes at 4°C.

[0014] In a further application embodiment, the centrifugation condition in step (4) is centrifugation at 17000×g for 15 minutes at 4°C.

[0015] In a further application embodiment, the vitrification cryopreservation solution in step (5) contains 15% DMSO, 15% ethylene glycol, 20% dextran and 10% trehalose, and the balance is DMEM culture medium.

[0016] In a further application embodiment, the vitrification cryopreservation in step (7) uses a programmable cryostat to control the cooling rate, and the sample cooling end point is set to -140°C.

[0017] The beneficial effects of the present invention are as follows: (1) The present invention uses keratinocyte exosomes to culture melanocytes, which can promote the proliferation of melanocytes. The increase in tyrosinase activity indicates that melanosome differentiation is more mature, melanin synthesis increases, and the increase in melanin granule content can lay a corresponding foundation for the clinical application of melanocytes in the treatment of vitiligo, premature graying of hair and other related diseases.

[0018] (2) The acquisition method provided by the present invention combines vitrification cryopreservation and a programmable cryostat, which can achieve aseptic cryopreservation, has a high cryopreservation cooling efficiency, causes less damage to melanosomes, is more convenient to apply, and does not require repeated material collection and culture. Description of the Drawings

[0019] Figure 1 is a transmission electron micrograph of the keratinocyte exosomes prepared in Example 1; Figure 2 is a bar graph of the particle size distribution of the keratinocyte exosomes prepared in Example 1; Figure 3 is a Western blot electrophoresis pattern of the keratinocyte exosomes prepared in Example 1; Figure 4 is a bar graph of the melanin granule concentration in each group in Example 2; Figure 5 is a cryopreservation cooling curve graph of each group in Example 3; Figure 6 is a bar graph of the melanin granule concentration in each group in Example 3; Figure 7 is a bar graph of the keratinocyte activity detected by CCK8 in each group in Example 3; Figure 8 It is a bar graph showing the expression levels of keratinocyte-related genes detected by qRT-PCR in each group in Example 3. Detailed implementation manners

[0020] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] Example 1: Preparation of keratinocyte-derived exosomes 1. Keratinocyte resuscitation Take 1 vial of P2-generation keratinocytes for resuscitation. Preheat the water bath to 37 °C in advance and pre-cool the centrifuge. Take out the cells from the liquid nitrogen tank, place them in an ice box and transport them to the side of the water bath. Put the cryopreservation tube into the water bath to thaw, transfer it to a 15 mL centrifuge tube, and centrifuge at 1300 rpm for 6 minutes. After centrifugation, aspirate and discard the supernatant, resuspend with 1 mL of keratinocyte medium, inoculate into a culture flask according to the required density, add 0.1 mmol / L ascorbic acid, shake well by the cross-shaking method, and then place it in an incubator for culture.

[0023] Preparation of keratinocyte medium: EpiLife® Medium + 1% human keratinocyte growth supplement HKGS, mix well.

[0024] 2. Isolation and purification of keratinocyte-derived exosomes (1) After normal culture of keratinocytes for 48 hours, collect the culture supernatant. The collected cell culture supernatant is filtered and sterilized through a 0.22 μm filter membrane.

[0025] (2) Centrifuge the supernatant at 2000×g for 30 minutes to remove sediment, dead cells and cell debris, and collect the supernatant; centrifuge the finally collected supernatant at 100000×g for 60 minutes, remove the supernatant to collect the precipitate, and resuspend the precipitate with a certain volume of PBS.

[0026] (3) Collect exosomes from the resuspended liquid through the EXODUS fully automatic exosome extraction system, and wash and resuspend and purify the extracted exosomes with a certain volume of PBS buffer (containing 2% trehalose, pre-cooled at 4 °C in advance). Aliquot the final product and store it temporarily in an ultra-low temperature refrigerator.

[0027] 3. Identification and detection of exosomes The identification technical solution is as follows: (1)Observation of the general morphology of exosomes by transmission electron microscopy: Prepare a slide of the keratinocyte exosome suspension and observe the morphology and size of the exosomes by transmission electron microscopy to preliminarily estimate the concentration; the results of transmission electron microscopy are as Figure 1 shown.

[0028] (2)Detection of particle size and concentration by nanoparticle tracking analyzer: Take the keratinocyte exosomes, thaw them in a water bath at 25 °C, and place them on ice. Dilute with PBS and directly use for NTA detection; the results of particle size detection are as Figure 2 shown. It can be seen that the particle size range of exosomes mainly concentrates between 55 and 100 nm, and the average particle size is 81.6 nm.

[0029] ③ Detection of the expression levels of CD81, CD63, TSG101, and CD9 proteins in exosomes by Western blot; the results of the expression of surface marker proteins are as Figure 3 shown.

[0030] Example 2: Cultivation of melanocytes 1. Resuscitation and culture of melanocytes Take 1 vial of P2-generation melanocytes for resuscitation. Preheat the water bath to 37 °C in advance, prepare an ice box with ice, and pre-cool the centrifuge. Take out the cells from the liquid nitrogen tank, place them in the ice box and transport them to the water bath. Put the cryopreservation tube into the water bath to thaw, transfer it to a 15 mL centrifuge tube, and centrifuge at 1300 rpm for 6 minutes. After centrifugation, aspirate and discard the supernatant, resuspend with DMEM complete medium containing 10% FBS, inoculate into a culture flask according to the required density, shake well and place it in the incubator for culture.

[0031] 2. Treatment of melanocytes Digest and count the P3-generation melanocytes, and adjust the cell concentration to 5×10 4 particles / mL according to the counting results, and inoculate into a 96-well plate (100 μL per well), with 3 replicates. Cell grouping and treatment: After the cells adhere overnight, discard the medium; in the experimental group, replace the medium with DMEM medium containing 1×10 8 particles / mL, 1×10 9 particles / mL, and 1×10 10 particles / mL respectively, and the control group is fresh DMEM medium, 100 μL per well; place it in a constant temperature incubator at 37 °C with 5% CO2 for incubation. The melanocytes in the 96-well plate are cultured with keratinocyte exosomes, and the melanin content is detected after 48 hours.

[0032] The detection method is as follows: (1)Take out the 96-well plate and observe the cell morphology under an inverted microscope; (2)After washing with PBS, add 500 µL of 0.25% trypsin to each well and digest for 1 minute; (3)After terminating the digestion, pipette the adherent cells and transfer them to a 2 mL EP tube. Centrifuge at 1800 rpm for 5 minutes and discard the supernatant; (4)Add 1 mL of 1 mol / L NaOH (containing 10% DMSO) solution to each tube and mix well; (5)Incubate at 60 °C for 1 hour; (6)Use an enzyme-linked immunosorbent assay detector to measure the OD value of each group at a wavelength of 470 nm.

[0033]

[0034] The relative melanin content measured after 48 hours of incubation is as Figure 4 shown. Adding keratinocyte exosomes in the experimental group can increase the melanin content. Among them, when the concentration of keratinocyte exosomes is 1×10 10 particles / mL, the melanin content of the cells is significantly higher than that of the control group, and the melanin content increases by 52.7%.

[0035] 3. Isolation and purification of melanin granules Digest and count the P3 generation melanocytes. According to the counting results, adjust the cell concentration to 5×10 4 / mL. Add DMEM medium containing 1×10 10 particles / mL of keratinocyte exosomes and inoculate them into a T175 culture flask. After incubating for 48 hours, collect the melanocyte culture supernatant. Centrifuge at 2000×g for 10 minutes at 4 °C, aspirate and discard the supernatant. Resuspend the precipitate with 1 mL of PBS. Filter the resuspended melanin granule suspension through a 20 μm filter membrane to remove suspended melanocytes and large impurity particles in the medium itself. Centrifuge the filtered melanin granule suspension at 17000×g for 15 minutes at 4 °C. Aspirate and discard the supernatant. Cover the precipitate with PBS to prevent drying and store it at -20 °C.

[0036] Example 3: Control experiment on the freezing method of melanin granules (1)Take the melanin granules prepared in Example 2 and set up a control group, experimental group A and experimental group B respectively to verify the differences in different freezing methods. In the attached figure, "Con" is marked as the control group, "A" is experimental group A, and "B" is experimental group B.

[0037] For the melanin granules in the control group, add commercially available conventional cryopreservation solution, place them in a programmable cooling box, and transfer them to a liquid nitrogen tank after placing them at -80 °C for more than 2 hours.

[0038] The experimental group A was subjected to improved vitrification cryopreservation treatment: ① Pretreatment: Melanin granules were added to 4 mL of vitrification cryopreservation solution diluted 2-fold with culture medium and pre-equilibrated for 2 minutes. After centrifuging to remove the supernatant, vitrification cryopreservation solution was quickly added and aliquoted into 2-mL cryotubes, 500 μL per tube, and placed in a 4-degree pre-cooled environment for 2 minutes.

[0039] The formula of the vitrification cryopreservation solution was 15% DMSO + 15% ethylene glycol + 20% dextran + 10% trehalose.

[0040] ② Adjust the parameters of the programmable freezer: The program was as follows: ① -25°C / min to -140°C; 2. holding at -140°C. This program ran continuously, and the data of the thermocouple was intercepted by recording the intermediate time points. The end point of sample cooling: -140°C.

[0041] ③ Execute the above program to complete the cooling of the programmable freezer cavity, then place the cryotubes into a foam box filled with liquid nitrogen. The liquid nitrogen covered half of the cryotubes but did not cover the tube mouths, and the foam box was quickly placed into the programmable freezer. Ensure that one of the tubes in the vitrification cryopreservation solution was inserted with a thermocouple, and the rest were sealed.

[0042] ④ The cryotubes were cooled simultaneously. After the cooling was completed (when the reading of the thermocouple reached -90°C), the cryotubes were visually inspected with a lamp inspection instrument to check whether the liquid in the cryotubes was in a typical vitrified state, and the cooling rate was recorded. After the cryotubes showed a vitrified state, they were transferred to a liquid nitrogen tank.

[0043] The experimental group B was subjected to ordinary vitrification cryopreservation treatment: The melanin granules were placed on the sample plate of the vitrification perfusion device, and vitrification cryopreservation solution DAP213 and vitrification cryopreservation buffer were added respectively. After perfusion, the sample plate was quickly placed into a vitrification refrigeration container, the temperature and vitrification formation time were recorded, and after the sample showed a vitrified state, it was transferred to a liquid nitrogen tank.

[0044] The cooling curves of the three groups are as Figure 5 shown. The control group samples took 110 minutes to cool to -80°C, with a slower cooling rate, and ice crystal formation caused certain damage to the samples. Compared with the experimental group B, the experimental group A could cool to -140°C within 10 minutes, and the cooling rate was significantly better than that of group B, indicating that adopting a specific combination of vitrification cryopreservation solution and instrument could reduce ice crystal formation, improve the cooling rate, and better protect the cryopreserved particles.

[0045] (2) Thawing and removal of the cryopreservation solution: Take out 3 groups of melanin granules and immediately put them into a 37°C constant temperature water bath for rapid thawing. After thawing, add 3 times the volume of PBS to the melanin granules to centrifuge and remove the solution, wash twice with PBS repeatedly, and then detect the content of melanin granules according to the method provided in Example 2.

[0046] The detection results are as Figure 6 shown. After the control group was slowly frozen using ordinary programming, the preservation effect of melanin was poor, and the content after resuscitation was low. The effects of Group A and Group B were better than those of the control group, and the melanin content after resuscitation in Group A could be increased by more than one time compared with the control, proving that it has an excellent effect on preserving melanin granules.

[0047] (3)Effect of resuscitated melanin granules on the activity of keratinocytes: Take out the 96-well plate and observe the cell morphology under an inverted microscope; aspirate the culture medium in each well, wash twice with PBS, add 10 μL of the prepared CCK-8 and 90 μL of DMEM mixture to each well, pay attention not to generate bubbles, and incubate in a 37°C constant temperature incubator in the dark for 1 hour; use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance (OD) value (at a wavelength of 450 nm) of each well.

[0048]

[0049] CCK-8 was used to detect the cell proliferation activity, and the detection results are as Figure 7 shown. The cell proliferation activities of keratinocytes supplemented with melanin granules from Group A and Group B were significantly increased compared with the control; the cell proliferation activity of keratinocytes supplemented with melanin granules from Group A was significantly higher than that of Group B.

[0050] (4)Effect of resuscitated melanin granules on the expression levels of genes related to keratinocytes To further explore the effect of melanin granules on keratinocytes, use the Quic-Start ProtocolRNeasy@ Mini Kit (74104) kit to extract the RNA of keratinocytes, reverse transcribe the cDNA through the TaqMan TM MicroRNA reverse transcription kit (Thermo Fisher Scientific), and finally use the Takara TB Green kit to perform the reaction through a fluorescence quantitative PCR instrument to obtain the relative expression levels of keratinocyte stemness genes (KRT14, KRT5, KRT15) and differentiation genes (KRT1, KRT10).

[0051] The results are as Figure 8As shown. Compared with the control, the stem cell genes (KRT5, KRT15) of keratinocytes in group A were up-regulated, indicating that keratinocytes treated with group A had stronger proliferation potential on the surface. It has been found that the differentiation of keratinocytes inhibits the proliferation of keratinocytes. Compared with the control, the expression of the differentiation genes KRT1 and KRT10 was decreased, suggesting that adding melanin granules cryopreserved by specific vitrification treatment can better promote the proliferation of keratinocytes.

[0052] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description, and thus it is intended to embrace all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A method for obtaining human-derived melanin granules, characterized in that, Including the steps: (1) Cultivate melanocytes to the P3 generation, inoculate them into a culture vessel, and replace the culture medium with DMEM medium containing keratinocyte exosomes after the cells adhere to the wall, and incubate for at least 48 hours; (2) Collect the culture supernatant, aspirate and discard the upper layer of the supernatant after centrifugation, and resuspend the precipitate with PBS; (3) Filter with a 20 μm filter membrane to remove the filter residue; (4) Aspirate and discard the upper layer of the supernatant after centrifuging the filtrate.

2. The method for obtaining melanin granules according to claim 1, wherein The preparation method of the keratinocyte exosome-containing product includes the steps: (a) Recover the culture supernatant after culturing keratinocytes for at least 48 hours, and filter and sterilize it with a 20 μm filter membrane; (b) After centrifuging at 2000×g for 30 minutes, retain the supernatant, then after centrifuging at 100000×g for 60 minutes, retain the precipitate and resuspend it with PBS solution; (c) Extract the exosomes from the collected suspension.

3. The method for obtaining melanin particles according to claim 2, wherein The culture medium used in step (a) is EpiLife® Medium serum-free medium supplemented with 1% human keratinocyte growth supplement HKGS and 0.1 mmol / L ascorbic acid.

4. The method for obtaining melanin particles according to any one of claims 1 to 3, characterized in that, The concentration of keratinocyte exosomes in the DMEM medium containing keratinocyte exosomes described in step (1) is 1×10 8 ~1×10 10 particles / mL.

5. The method for obtaining melanin granules according to claim 1, wherein The centrifugation conditions in step (2) are centrifugation at 2000×g for 10 minutes at 4°C.

6. The method for obtaining melanin granules according to claim 1, wherein, The centrifugation conditions in step (4) are centrifugation at 17000×g for 15 minutes at 4°C.

7. The method for obtaining melanin granules according to claim 1, wherein, Also including the steps: (5) Add vitrification cryopreservation solution diluted with the culture medium for pre-equilibration; (6) Aspirate and discard the upper layer of the supernatant after centrifugation, and then transfer it to a cryotube for pre-cooling at 4°C; (7) Perform vitrification cryopreservation at a rate of decreasing 25°C per minute, and store it in a liquid nitrogen environment after completion.

8. The method for obtaining melanin granules according to claim 7, wherein The vitrification cryopreservation solution in step (5) contains 15% DMSO, 15% ethylene glycol, 20% dextran, and 10% trehalose, and the balance is DMEM culture solution.

9. The method for obtaining melanin particles according to claim 7, wherein, The vitrification cryopreservation in step (7) uses a programmable freezer to control the cooling rate, and sets the cooling end point to -140°C.

10. The method for obtaining melanin granules according to claim 7, wherein Also including the steps: (8) Take out the cryotube in the liquid nitrogen environment, thaw it in a 37°C constant temperature water bath, add 3 volumes of PBS solution for washing after thawing, centrifuge to remove the upper layer of the supernatant, and repeat the washing at least 2 times.

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