Construction method of mouse hair follicle organoid

Abstract

The invention discloses a construction method of mouse hair follicle organoid, and relates to the technical field of biology, and the key point of the technical scheme is that the construction method comprises the following steps: obtaining back skin of a mouse, cutting into pieces, digesting, blowing, beating and releasing, filtering and collecting, removing blood cells, re-suspending and counting, preparing a cell suspension, planking, carrying out centrifugal treatment, and culturing. According to the invention, the source of mice, namely the source of seed cells, is expanded, the operation is simple and convenient, matrigel does not need to be used, and the cost is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for constructing mouse hair follicle organoids. Background Art

[0002] There are very limited methods to treat hair loss. To date, the only FDA-approved treatments are minoxidil and finasteride. However, the effects of these drugs are unsatisfactory, and the treatment takes a long time to take effect. Symptoms recur after discontinuation of the drug.

[0003] Compared to traditional two-dimensional cells, organoids have a three-dimensional structure similar to real organs, contain multiple cell types, and can partially reproduce organ functions. Organoids have important applications in biomedical research, drug development, disease modeling, and other fields. Organoids offer significant advantages as novel disease models. Compared to two-dimensional cultures, organoids can not only be cultured for long periods of time but also have stable phenotypic and genetic characteristics, making them amenable to genome sequencing and expression profiling.

[0004] The existing technology is to separate the dermis and epidermis of the dorsal skin of pregnant mice at 18.5 (E18.5) and then dissociate them into single cells. The two are mixed in vitro, and the mixture is formed into cell spheres using Matrigel. Organoids containing hair follicles are formed in vitro. However, this method is limited to the use of pregnant mice to construct hair follicle organoids. The operation is complicated and requires the use of Matrigel, which is an extract from mouse tumors and has certain risks in industrial applications. The use of Matrigel needs to be operated at low temperatures, is easy to solidify during the operation, and is expensive. In view of this, the present invention proposes a method for constructing mouse hair follicle organoids. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing mouse hair follicle organoids to solve the above problems.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A method for constructing mouse hair follicle organoids, comprising the following steps:

[0007] The back skin of the mouse was obtained, minced, digested, released by pipetting, collected by filtration, blood cells removed, resuspended and counted, a cell suspension was prepared, plated, centrifuged, and cultured.

[0008] The present invention is further configured such that: the mouse is an embryonic mouse or a mouse after birth.

[0009] The present invention is further configured as follows: the shredding is shredding with scissors to 3-4 mm 2 .

[0010] The present invention is further configured as follows: the digestion uses 2 mg / ml collagenase A as a digestive enzyme and is performed at 37° for 40-60 minutes.

[0011] The present invention is further configured as follows: the blowing release is: the digested tissue cells are blown with a Pasteur pipe to release the cells, then 1 ml of a culture medium containing 10% serum is added, the cells are blown again 8-12 times, and 5 ml of a DMEM culture medium is added.

[0012] The present invention is further configured as follows: the cell collection is: filtering through a 40um cell sieve, taking the cell fluid, and transferring it to a 15ml centrifuge tube, centrifuging at 500g for 5 minutes, removing the supernatant, and collecting the cell precipitate.

[0013] The present invention is further configured as follows: the blood cell removal is as follows: the collected cells contain visible red blood cell clumps, red blood cell lysis solution is added to blow the cell sediment, after standing at room temperature for 1-3 minutes, DMEM is added, centrifuged at 500g for 5 minutes, and the sediment is collected.

[0014] The present invention is further configured as follows: the cell culture medium is evenly spread in a low-adsorption 96-well culture plate with 15,000 cells per well and 200 μl of culture medium per well.

[0015] The present invention is further configured as follows: the centrifugation treatment is to centrifuge the 96-well culture plate at 300g for 5 minutes using a horizontal centrifuge.

[0016] The present invention is further configured as follows: the culture conditions are: 37°, 5% CO2 concentration.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. No need to separate the true epidermis. In our method, the mouse dorsal skin is directly digested with 2mg / ml collagenase A to obtain a mixed single-cell suspension. This simplifies the operation steps and does not require true epidermal separation. The dorsal skin can be directly digested, shortening the experimental time. It also expands the source of mice, that is, the source of seed cells, which can come from mice in the late pregnancy, E18.5-19.5, or from the dorsal skin of newborn mice P0-P1.

[0019] 2. Directly plate the cells obtained in step 2 into a low-adsorption 96-well plate and centrifuge them at 300g for 3 minutes using a horizontal centrifuge to aggregate the cells. Cell spheres will form the next day, and hair follicles will be produced on the 4th day, forming organoids containing hair follicles, avoiding the use of Matrigel. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Schematic diagram of light microscopic photographing results and HE staining of the method before and after improvement of the present invention;

[0021] Figure 2 Schematic diagram of immunofluorescence staining of the method before and after improvement of the present invention. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Example 1:

[0024] A method for constructing mouse hair follicle organoids comprises the following steps:

[0025] 1. Obtaining Mouse Dorsal Skin: Pregnant mice at E18.5 / E19.5 were sacrificed by cervical dislocation, with the abdomen facing upward. After disinfection with alcohol spray, the abdomen was cut open with sterile scissors to expose the embryo. The uterus was carefully opened and the embryo was removed and placed in a new dish. After decapitation, the dorsal skin (from the upper and lower limbs) was removed with scissors.

[0026] 2. Cut the back skin into small pieces: Use scissors to cut the back skin tissue obtained above into small pieces of about 3-4mm 2 ;

[0027] 3. Digestion: Add 1 ml of 2 mg / ml collagenase A to the minced tissue and digest at 37°C for approximately 40-60 minutes, depending on the size of the mouse.

[0028] 4. Release cells: Use a Pasteur pipette to pipette the digested tissue to release the cells, then add 1 ml of culture medium containing 10% serum, pipette again about 10 times, and add 5 ml of DMEM medium;

[0029] 5. Cell collection: Filter the liquid containing tissues and cells through a 40 μm cell sieve and transfer to a 15 ml centrifuge tube. Centrifuge at 500 g for 5 minutes, remove the supernatant, and collect the cells.

[0030] 6. Remove blood cells: The collected cells may contain red blood cell clumps. Add 1 ml of red blood cell lysis buffer to the cell pellet. After standing at room temperature for 2 minutes, add 9 ml of DMEM and centrifuge at 500 g for 5 minutes to collect the pellet.

[0031] 7. Count: Resuspend the cells from step 6 in 1 ml of complete culture medium and count;

[0032] 8. Plating: Use 15,000 cells / well to calculate the required cell volume. Prepare a cell suspension based on a liquid volume of 200 μl per well. Add the cell suspension to a 50 ml sample reservoir and add the sample using a dispenser.

[0033] 9. Centrifugation: After adding the plate in step 8, balance it and centrifuge it at 300g for 5 minutes in a horizontal centrifuge to aggregate the cells.

[0034] 10. Culture: Place the centrifuged plate in an incubator at 37°C and 5% CO2 for culture, performing a half-medium change every other day.

[0035] The above complete culture medium: DMEM / F12+1% Glutamax+1% PS.

[0036] Example 2:

[0037] The difference from Example 1 is that in step 1, newborn mice P0-P1 are used. After the mice are decapitated, the back skin is disinfected with an alcohol cotton ball, and the back skin (from both ends of the upper limbs to both ends of the lower limbs) is removed with scissors.

[0038] Comparative Example 1:

[0039] Pregnant mice at E18.5 were used. After obtaining embryonic mice, the dorsal skin was removed and digested with Dispase II at 4 degrees for 1 hour to separate the dermis and epidermis. The dermis and epidermis were then digested with collagenase I at 37 degrees for 80 minutes, and the epidermal cells were then digested with trypsin at 37 degrees for 10 minutes to obtain single-cell suspensions of the dermis and epidermis. After filtration, epithelial and dermal cells were obtained, counted separately, and then mixed in a 1:1 ratio. 2% Matrigel matrix gel was added to complete culture medium, and the cell suspension was added to the plate at a density of 20,000 cells per well. The plate was then placed in a 4-degree refrigerator for more than 30 minutes and then placed in an incubator for culture.

[0040] The results of hair follicle organoid culture are as follows Figure 1 and Figure 2 As shown, Comparative Example 1 is before improvement, and Example 1 and Example 2 are after improvement. Before and after improvement, it can be seen that the hair follicle organoids are successfully constructed, and multiple hair follicles grow in the organoids.

[0041] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for constructing mouse hair follicle organoids, characterized by: The steps include: The back skin of the mouse was obtained, minced, digested, released by pipetting, collected by filtration, blood cells removed, resuspended and counted, plated, centrifuged, and cultured.

2. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The mouse is an embryonic mouse or a postnatal mouse.

3. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The cutting is to cut into pieces of 3-4 mm using scissors. 2 .

4. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The digestion was performed using 2 mg / ml collagenase A as a digestion enzyme at 37° for 40-60 minutes.

5. The method for constructing mouse hair follicle organoids according to claim 4, wherein: The blowing release is as follows: the digested tissue cells are blown with a Pasteur pipette to release the cells, and then 1 ml of a culture medium containing 10% serum is added, and the cells are blown again 8-12 times, and 5 ml of a DMEM culture medium is added.

6. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The cells were collected by filtering through a 40 μm cell sieve, taking the cell solution, transferring it to a 15 ml centrifuge tube, centrifuging at 500 g for 5 minutes, removing the supernatant, and collecting the cell pellet.

7. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The blood cell removal is as follows: the collected cells contain red blood cell clumps, red blood cell lysis solution is added to blow the cell pellet, after standing at room temperature for 1-3 minutes, DMEM is added, centrifuged at 500g for 5 minutes, and the pellet is collected.

8. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The cell culture medium was evenly plated in a 96-well culture plate with low adsorption, with 15,000 cells per well and 200 μl of culture medium per well.

9. The method for constructing mouse hair follicle organoids according to claim 1, wherein: The centrifugation treatment is to centrifuge the 96-well culture plate at 300 g for 5 minutes using a horizontal centrifuge.

10. The method for constructing mouse hair follicle organoids according to claim 9, wherein: The culture conditions are: 37°C, 5% CO2.

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