Preparation method of mouse skin organoid

Abstract

The invention discloses a preparation method of a mouse skin organoid, which comprises the following steps: separating dermis and epidermis of an E18.5 mouse to obtain dermis cells, mixing the dermis cells with back skin cells of an E14.5 embryo mouse, mixing by using Matrigel to form cell spheres, and gradually forming the skin organoid in in-vitro culture; the organoid is mainly applied to batch production of organoid containing hair follicles. The invention relates to the technical field of stem cells and regenerative medicine. According to the preparation method of the mouse skin organoid, a cell source is improved in the experiment process, the mouse skin organoid containing hair follicles can still be formed after cells in the early development stage are added, and related developmental biology research is carried out. And the source of another dermal cell is not limited to late pregnancy, and also can be a newborn mouse, so that the source of seed cells is expanded, and the cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells and regenerative medicine, and specifically to a method for preparing mouse skin organoids. Background Art

[0002] Previous literature reports used pregnant mice at E18.5. After obtaining the 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 type I collagenase at 37 degrees for 80 minutes, and the epidermal cells were digested with 0.25% trypsin at 37 degrees for 10 minutes to obtain single-cell suspensions of the dermis and epidermis. After filtration, the epithelial and dermal cells were obtained, counted, and mixed in a 1:1 ratio. 2% Matrigel was added to complete culture medium, and the cell suspension was added to a plate at a density of 20,000 cells per well. The plate was then placed in a refrigerator at 4 degrees for at least 30 minutes and then placed in an incubator for culture. However, the production of these organoids used dorsal skin tissue from late-pregnant mice as the cell source, many of which were at a late stage of development, which limited the research related to developmental biology.

[0003] Reference publication number CN117625514B discloses an artificial hair follicle, its in vitro construction method, and its application. Using the skin organoid technology of the present invention, a single hair follicle can be transformed into 20-30 artificial hair follicles, a 20-30-fold increase in the original number. Cells derived from a single initial hair follicle, after treatment with the steps of the present invention, can form 20-30 artificial hair follicle spheres under optimized conditions. This technology addresses the technical and medical bottlenecks that currently hinder the expansion of hair follicles through traditional hair follicle transplantation techniques.

[0004] The existing technology mainly uses the back skin cells of E18.5 mice to dissociate the dermis and epidermis into single cells, mix them, and use Matrigel to form cell spheres. In vitro culture gradually forms hair follicle organoids; this organoid is mainly used to mass produce organoids containing hair follicles, and has not yet been applied. The flow chart is as follows Figure 1 As shown, the organoids formed by existing technology are composed of skin cells of mice in the late developmental stage, i.e., E18.5, and lack cells of early development. Their applications are limited, and it is difficult to conduct research related to cell development.

[0005] The present invention improves the cell source during the experimental process, adds cells in the early stages of development, and can still form mouse skin organoids containing hair follicles, and conduct related developmental research.

[0006] 1. After isolating the epidermis of E18.5 mice, the dermal cells were taken and combined with the dorsal skin cells of E14.5 mice to form organoids containing developmental mouse skin cells for skin development research.

[0007] 2. In vitro small molecule experiments were conducted on these organoids. The organoids composed of E18.5 true epidermis had no response to the small molecule, while the organoids composed of E14.5 skin cells and E18.5 dermal cells responded sensitively to it. Summary of the Invention

[0008] (1) Technical problems solved

[0009] To address the shortcomings of existing technologies, the present invention provides a method for preparing mouse skin organoids. Cells derived from the dorsal skin tissue of early-stage mice are incorporated into the organoid system to study the influence of developmental signals on cell fate. Furthermore, the source of the additional dermal cells is not limited to late-gestation mice; it can also be obtained from newborn mice, expanding the seed cell source and reducing costs.

[0010] (2) Technical solution

[0011] To achieve the above objectives, the present invention is implemented through the following technical solutions: a method for preparing mouse skin organoids, using dermal cells from E18.5 mice and dorsal skin cells from E14.5 embryonic mice, which are mixed and then mixed using Matrigel to form cell spheres, and then gradually form skin organoids during in vitro culture. The organoids are used to batch produce organoids containing hair follicles. The method for obtaining dermal cells from the dorsal skin of pregnant E18.5 embryonic mice or newborn mice specifically includes the following steps:

[0012] S1. Obtaining the dorsal dermis of pregnant mice at E18.5 or newborn mice: After cervical dislocation, the pregnant mice were killed with the abdomen facing upwards and disinfected with alcohol spray. The abdomen was cut open with sterile scissors to expose the embryos. The uterus was carefully opened and the embryos were removed and placed in a new dish. After decapitation, the dorsal skin was removed with scissors. After P0-P1 mice were killed by decapitation, the dorsal skin was disinfected with alcohol cotton balls and removed with scissors. The dorsal skin was then washed with sterile PBS.

[0013] S2. Separation of dermis and epidermis: Cut the mouse back skin tissue removed in step S1 into pieces of about 5 mm * 5 mm in size and place it in a sterile dish with the dermis facing down. Flatten it and add 1 ml of Dispase to cover the skin tissue. Place it in a 37°C incubator for digestion for 1-2 hours, depending on the hardness of the skin.

[0014] S3. After the tissue digestion in step S2 is completed, the dermis and epidermis are separated. The epidermis is discarded after separation, and the dermis tissue block is transferred to a new dish and minced with ophthalmic scissors 10-20 times;

[0015] S4. Digestion of dermal tissue: Add 1 ml of 2 mg / ml collagenase A to the minced tissue and digest at 37°C for 40-60 minutes.

[0016] S5. Obtaining dermal cells: The digested tissue was pipetted 10 times with a Pasteur pipette to release the cells. A large number of cells were observed under a microscope. Subsequently, 1 ml of culture medium containing 10% serum was added and pipetted again 10 times. Finally, 5 ml of DMEM culture medium was added. The liquid containing the tissue and cells was filtered through a 40 μm cell sieve and transferred to a 15 ml centrifuge tube. The cells were centrifuged at 500 g for 5 minutes to collect the cells. If the collected cells contained red blood cells, the red blood cells were removed with red blood cell lysis buffer and the cells were collected again. That is, the cell pellet was pipetted with 1 ml of red blood cell lysis buffer, allowed to stand at room temperature for 2 minutes, and then 9 ml of DMEM was added and centrifuged at 500 g for 5 minutes.

[0017] S6. Resuspend the dermal cells of E18.5 newborn mice obtained in step S5 in complete culture medium and count them for later use.

[0018] Preferably, in step S1, the back skin is removed from both ends of the upper limbs to both ends of the lower limbs using scissors.

[0019] Preferably, when separating the dermis and the epidermis in step S3, the tweezers can be used to gently clamp a corner of the epidermis to easily separate it from the dermis.

[0020] Preferably, the method for obtaining skin cells from the back of an E14.5 embryonic mouse comprises the following steps:

[0021] T1, dorsal skin of pregnant mice at E14.5: After the pregnant mice were killed by cervical dislocation, the abdomen was facing upwards and disinfected 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 was gently picked up with forceps under a microscope and placed in a new sterile dish.

[0022] T2. Digest tissue to obtain cells: Cut the E14.5 dorsal skin tissue from step T1 into small pieces 10-20 times. Add 1 ml of 2 mg / ml collagenase A to the tissue and digest at 37°C for approximately 1 hour. Then remove the tissue and pipette 10 times with a Pasteur pipette. Collect cells and remove red blood cells as in step S5.

[0023] T3. Resuspend the E14.5 embryonic mouse dorsal skin cells obtained in step T2 in complete culture medium and count them for later use;

[0024] T4. The cells obtained in step S6 and step T3 were mixed at a ratio of E14.5ds:E18.5-P0 M=1:1, with a total cell volume of 20,000 cells per well;

[0025] T5. Pre-cool the culture medium: Advanced DMEM / F12 + 1% Glutamax + 1% PS + 1.5% Matrigel.

[0026] Preferably, in step T1, the back skin is gently pinched from both ends of the upper limb to both ends of the lower limb using tweezers under a microscope and placed in a new sterile dish.

[0027] Preferably, the cells mixed in step T4 are calculated with a liquid volume of 200 ul per well to prepare a cell suspension, and the cell suspension is added to a 50 ml sample loading tank. The sample is loaded with a pistol, and the pistol tip, sample loading tank, and culture plate are pre-cooled in advance and operated on ice. The plate is then placed in a 4 degree refrigerator and incubated for 30-60 minutes.

[0028] Preferably, the centrifuge is pre-cooled to 4 degrees in advance, the plate is taken out, leveled, and centrifuged at 300g for 5 minutes using a horizontal centrifuge to aggregate the cells.

[0029] Preferably, the centrifuged plate is placed in an incubator at 37 degrees Celsius and 5% CO2 for culture, and half of the medium is changed every other day. If small molecules are added, they are added to the culture medium during the half-medium change on the second day, and the drug concentration at this time is set to twice the concentration.

[0030] (3) Beneficial effects

[0031] The present invention provides a method for preparing mouse skin organoids. Compared with existing technologies, the method has the following advantages: the mouse skin organoid preparation method combines dermal cells from E18.5 mice with dorsal skin cells from E14.5 mice, and then gradually forms skin organoids during in vitro culture; the organoids are mainly used to mass-produce organoids containing hair follicles. During the experimental process, the present invention improves the cell source, adding cells from early developmental stages, and still allows the formation of mouse skin organoids containing hair follicles, and conducts research related to skin development. In vitro small molecule experiments were conducted on these organoids. Organoids composed of E18.5 epidermis did not respond to the small molecule, while organoids composed of E14.5 skin cells and E18.5 dermal cells responded sensitively. This indicates that by adding cells derived from the dorsal skin tissue of early-stage mice to the organoid system, it can be used to study the influence of developmental signals on cell fate. Moreover, the source of the other dermal cells is not limited to late pregnancy; it can also be newborn mice, which expands the source of seed cells and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flowchart for constructing mouse skin organoids from E18.5 cells;

[0033] Figure 2Flowchart of the mouse skin organoids formed by assembling E14.5 skin cells and E18.5 dermal cells of the present invention;

[0034] Figure 3 This is a graph showing no significant changes in MCs in E18.5-derived mouse skin organoids after PRCi treatment of the present invention;

[0035] Figure 4 This figure shows a significant increase in MCs in mouse skin organoids composed of E14.5ds / E18.5 after PRCi treatment of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] See also Figure 1-4 The present invention provides two technical solutions: a method for preparing mouse skin organoids, specifically including the following embodiments:

[0038] Example 1: A method for preparing mouse skin organoids, using dermal cells from E18.5 mice and dorsal skin cells from E14.5 embryonic mice, which are mixed and then gradually formed into skin organoids in in vitro culture. The organoids are used to batch produce organoids containing hair follicles. The method for obtaining dermal cells from the dorsal skin of pregnant E18.5 embryonic mice or newborn mice specifically includes the following steps:

[0039] S1. Obtaining the dorsal dermis of pregnant mice at E18.5 or newborn mice: Pregnant mice at E18.5 / E19.5 or newborn mice at P0-P1 can be used. Pregnant mice were killed 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 was removed with scissors. For P0-P1 mice, the dorsal skin was disinfected with alcohol cotton balls and removed with scissors. The dorsal skin was then washed with sterile PBS.

[0040] S2. Separation of dermis and epidermis: Cut the mouse back skin tissue removed in step S1 into pieces of about 5 mm * 5 mm in size and place it in a sterile dish with the dermis facing down. Flatten it and add 1 ml of Dispase to cover the skin tissue. Place it in a 37°C incubator for digestion for 1-2 hours, depending on the hardness of the skin.

[0041] S3. After the tissue digestion in step S2 is completed, the dermis and epidermis are separated. The epidermis is discarded after separation, and the dermis tissue block is transferred to a new dish and minced with ophthalmic scissors 10-20 times;

[0042] S4. Digestion of dermal tissue: Add 1 ml of 2 mg / ml collagenase A to the minced tissue and digest at 37°C for 40-60 minutes.

[0043] S5. Obtaining dermal cells: The digested tissue was pipetted 10 times with a Pasteur pipette to release the cells. A large number of cells were observed under a microscope. Subsequently, 1 ml of culture medium containing 10% serum was added and pipetted again 10 times. Finally, 5 ml of DMEM culture medium was added. The liquid containing the tissue and cells was filtered through a 40 μm cell sieve and transferred to a 15 ml centrifuge tube. The cells were centrifuged at 500 g for 5 minutes to collect the cells. If the collected cells contained red blood cells, the red blood cells were removed with red blood cell lysis buffer and the cells were collected again. That is, the cell pellet was pipetted with 1 ml of red blood cell lysis buffer, allowed to stand at room temperature for 2 minutes, and then 9 ml of DMEM was added and centrifuged at 500 g for 5 minutes.

[0044] S6. Resuspend the dermal cells (Mesenchymal, M) of E18.5 newborn mice obtained in step S5 in complete culture medium and count them for later use.

[0045] In the embodiment of the present invention, in step S1, the back skin is removed from both ends of the upper limbs to both ends of the lower limbs using scissors.

[0046] In the embodiment of the present invention, when separating the dermis and the epidermis in step S3, the tweezers can be used to gently grasp a corner of the epidermis to easily separate it from the dermis.

[0047] Example 2: The technical solution of the embodiment of the present invention is different from that of Example 1 in that: a method for preparing mouse skin organoids, and a method for obtaining skin cells from the back of embryonic mice at E14.5 pregnancy specifically comprises the following steps:

[0048] T1, dorsal skin of pregnant mice at E14.5: After the pregnant mice were killed by cervical dislocation, the abdomen was facing upwards and disinfected 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 was gently picked up with forceps under a microscope and placed in a new sterile dish.

[0049] T2. Digest tissue to obtain cells: Cut the E14.5 dorsal skin tissue from step T1 into small pieces 10-20 times. Add 1 ml of 2 mg / ml collagenase A to the tissue and digest at 37°C for approximately 1 hour. Then remove the tissue and pipette 10 times with a Pasteur pipette. Collect cells and remove red blood cells as in step S5.

[0050] T3. Resuspend the E14.5 embryonic mouse dorsal skin cells (ds) in complete culture medium and count them for later use.

[0051] T4. The cells obtained in step S6 and step T3 were mixed at a ratio of E14.5ds:E18.5-P0 M=1:1, with a total cell volume of 20,000 cells per well;

[0052] T5, culture medium was pre-chilled in advance, Advanced DMEM / F12 + 1% Glutamax + 1% PS + 1.5% Matrigel;

[0053] T6. Plating: Prepare a cell suspension by adding 200 μl of liquid per well of the cells mixed in step T4. Add the cell suspension to a 50 ml sample loading tank and add the sample using a dispenser. Pre-cool the pipette tip, sample loading tank, and culture plate and operate on ice. Then, place the plate in a 4°C refrigerator and incubate for 30-60 minutes.

[0054] T7. Centrifugation: Pre-cool the centrifuge to 4 degrees Celsius, take out the plate, level it, and centrifuge it in a horizontal centrifuge at 300g for 5 minutes to aggregate the cells.

[0055] T8. Culture: Place the centrifuged plate in an incubator at 37°C and 5% CO2 for culture. Perform a half-medium change every other day. If small molecules are involved, add them to the culture medium during the half-medium change on the second day. At this time, the drug concentration can be set to twice the concentration.

[0056] In the embodiment of the present invention, in step T1, the back skin is gently pinched from both ends of the upper limb to both ends of the lower limb using tweezers under a microscope and placed in a new sterile dish.

[0057] The flowchart of mouse skin organoid construction and small molecule intervention involved in the present invention is as follows: Figure 2 After isolating the true epidermis of E18.5 mice, cells from the dermis were taken and combined with dorsal skin cells from E14.5 mice to form organoids containing developing mouse skin cells for use in skin development research.

[0058] The present invention conducted in vitro small molecule experiments on these organoids. According to research reports, this small molecule can promote the generation of Merkel cells (MCs) in the skin; the MC marker SOX2+K8+; PRCi has been reported to increase the number of MCs generated in mouse skin in vivo experiments. However, the MCs of skin organoids composed of E18.5 true epidermis did not change after the addition of PRCi, indicating that these organoids are not sensitive to PRCi ( Figure 3); however, after adding PRCi, the MC of skin organoids composed of E14.5 skin cells and E18.5 dermal cells increased significantly ( Figure 4 ), consistent with in vivo studies.

[0059] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0060] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0061] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing mouse skin organoids, characterized by: Dermal cells from E18.5 mice and dorsal skin cells from E14.5 embryonic mice are mixed and then mixed with Matrigel to form cell spheres. Skin organoids are gradually formed during in vitro culture. These organoids are used to mass-produce organoids containing hair follicles. The method for obtaining dermal cells from the dorsal skin of pregnant E18.5 embryonic mice or newborn mice specifically includes the following steps: S1. Obtaining dorsal skin from pregnant mice at E18.5 or newborns: Pregnant mice were killed by cervical dislocation, with the abdomen facing upward. The mice were disinfected with alcohol spray and the abdomen was cut open with sterile scissors to expose the embryos. The uterus was carefully opened and the embryos were removed and placed in a new dish. After decapitation, the dorsal skin was removed with scissors. P0-P1 mice were killed by decapitation, and the dorsal skin was disinfected with alcohol cotton balls. The dorsal skin was removed with scissors and washed with sterile PBS. S2. Separation of dermis and epidermis: Cut the mouse back skin tissue removed in step S1 into pieces of about 5 mm * 5 mm in size and place it in a sterile dish with the dermis facing down. Flatten it and add 1 ml of Dispase to cover the skin tissue. Place it in a 37°C incubator for digestion for 1-2 hours, depending on the hardness of the skin. S3. After the tissue digestion in step S2 is completed, the dermis and epidermis are separated. The epidermis is discarded after separation, and the dermis tissue block is transferred to a new dish and minced with ophthalmic scissors 10-20 times; S4. Digestion of dermal tissue: Add 1 ml of 2 mg / ml collagenase A to the minced tissue and digest at 37°C for 40-60 minutes. S5. Obtaining dermal cells: The digested tissue was pipetted 10 times with a Pasteur pipette to release the cells. A large number of cells were observed under a microscope. Subsequently, 1 ml of culture medium containing 10% serum was added and pipetted again 10 times. Finally, 5 ml of DMEM culture medium was added. The liquid containing the tissue and cells was filtered through a 40 μm cell sieve and transferred to a 15 ml centrifuge tube. The cells were centrifuged at 500 g for 5 minutes to collect the cells. If the collected cells contained red blood cells, the red blood cells were removed with red blood cell lysis buffer and the cells were collected again. That is, the cell pellet was pipetted with 1 ml of red blood cell lysis buffer, allowed to stand at room temperature for 2 minutes, and then 9 ml of DMEM was added and centrifuged at 500 g for 5 minutes. S6. Resuspend the dermal cells of E18.5 newborn mice obtained in step S5 in complete culture medium and count them for later use.

2. The method for preparing mouse skin organoids according to claim 1, wherein: In step S1, the back skin is removed from both ends of the upper limbs to both ends of the lower limbs using scissors.

3. The method for preparing mouse skin organoids according to claim 1, wherein: When separating the dermis and the epidermis in step S3, the tweezers can be used to gently grasp a corner of the epidermis to easily separate it from the dermis.

4. The method for preparing mouse skin organoids according to claim 1, wherein: The method for obtaining skin cells from the back of pregnant E14.5 embryonic mice specifically comprises the following steps: T1, dorsal skin of pregnant mice at E14.5: After the pregnant mice were killed by cervical dislocation, the abdomen was facing upwards and disinfected 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 was gently picked up with forceps under a microscope and placed in a new sterile dish. T2. Digest tissue to obtain cells: Cut the E14.5 dorsal skin tissue from step T1 into small pieces 10-20 times. Add 1 ml of 2 mg / ml collagenase A to the tissue and digest at 37°C for approximately 1 hour. Then remove the tissue and pipette 10 times with a Pasteur pipette. Collect cells and remove red blood cells as in step S5. T3. Resuspend the E14.5 embryonic mouse dorsal skin cells obtained in step T2 in complete culture medium and count them for later use; T4. The cells obtained in step S6 and step T3 were mixed at a ratio of E14.5ds:E18.5-P0 M=1:1, with a total cell volume of 20,000 cells per well; T5. Pre-cool the culture medium: Advanced DMEM / F12 + 1% Glutamax + 1% PS + 1.5% Matrigel.

5. The method for preparing mouse skin organoids according to claim 4, wherein: In step T1, the back skin is gently pinched from both ends of the upper limb to both ends of the lower limb using tweezers under a microscope and placed in a new sterile dish.

6. The method for preparing mouse skin organoids according to claim 4, wherein: The cells mixed in step T4 were added to a liquid volume of 200 μl per well to prepare a cell suspension, which was added to a 50 ml sample loading tank. Samples were loaded using a dispenser. The tip, sample loading tank, and culture dish were pre-cooled and operated on ice. The plate was then placed in a 4°C refrigerator and incubated for 30-60 minutes.

7. The method for preparing mouse skin organoids according to claim 4, wherein: Precool the centrifuge to 4 degrees in advance, take out the plate, level it, and centrifuge it in a horizontal centrifuge at 300g for 5 minutes to aggregate the cells.

8. The method for preparing mouse skin organoids according to claim 4, wherein: Place the centrifuged plate in an incubator at 37 degrees and 5% CO2 for culture, and perform half-medium change every other day. If small molecule drugs are involved, add them to the culture medium during half-medium change on the second day. At this time, the drug concentration can be set to twice the concentration.

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