CN116024159A - Method for constructing murine skin organoids - Google Patents
Method for constructing murine skin organoids Download PDFInfo
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- CN116024159A CN116024159A CN202211653817.XA CN202211653817A CN116024159A CN 116024159 A CN116024159 A CN 116024159A CN 202211653817 A CN202211653817 A CN 202211653817A CN 116024159 A CN116024159 A CN 116024159A
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Abstract
The invention relates to the technical field of organoids, and discloses a method for constructing a murine skin organoid. The method comprises the steps of placing murine skin primary cells into a murine skin organoid culture medium for organoid culture, wherein the murine skin organoid culture medium contains nicotinamide, dexamethasone and Y-27632. The method can realize efficient construction, passage and amplification of the murine skin organoids. Moreover, by adopting the preferred culture medium, the same culture medium can be adopted in the construction, amplification and passage processes of the murine skin organoids, so that the experimental process is greatly simplified, and the possibility of experimental errors caused by the need of using different culture mediums at different stages is greatly reduced.
Description
Technical Field
The invention relates to the technical field of organoids, in particular to a method for constructing a murine skin organoid.
Background
Organoids belong to three-dimensional (3D) cell cultures, have histological features that are highly similar to the corresponding human organs, and reproduce part of the physiological functions of the organs. These 3D systems replicate the complex spatial morphology of differentiated tissues and can exhibit cell-to-cell and cell-to-matrix interactions. Organoids remove confounding variables that animal models may introduce, but provide a higher degree of complexity than homogenized 2D cell cultures. By combining a high level of physiological relevance with the cheapness of in vitro procedures, organoids have the potential in many cases to aid or replace in vitro use of primary cells or immortalized cell lines and animal experiments. In addition, organoids have a high degree of genetic stability in culture, maintaining the genotype and phenotype of the tissue from which they are derived. Therefore, the organoids have wide application prospects in various fields such as stem cells and development, regenerative medicine, disease research, drug development, accurate medicine and the like.
Skin is the largest organ of human body, the types of skin diseases are various, the causes of the skin diseases are many, and a plurality of visceral diseases are correspondingly characterized and reflected on the skin, so that animal models are difficult to provide accurate conclusions on the research of the skin diseases due to a plurality of confusion variables. Moreover, many dermatological disorders, such as those caused by infectious agents, such as leprosy, scabies, mycoses, skin bacterial infections, etc., are often contagious, not only affecting physical health, but also causing panic and social discrimination. At present, researchers adopt human skin tissues (such as skin tissues provided by a patient) to conduct organoid culture, and conduct targeted treatment scheme research, drug research and the like. However, there are few ways to obtain human samples and there are many ethical restrictions on the use of human samples, so the development of skin organoids of animal origin can provide more convenience for the development of research works. However, there is currently no report on any method of constructing animal skin organoids.
Disclosure of Invention
The invention aims to solve the problems that in the prior art, the source of samples is less in the construction process of human skin organoids, and the application process is more restricted by ethics, so that the development of research work is limited, and the like.
In order to achieve the above object, the present invention provides a method for constructing a murine skin organoid, said method comprising placing murine skin primary cells in a murine skin organoid culture medium for organoid culture, wherein said murine skin organoid culture medium comprises nicotinamide, dexamethasone and Y27632.
In a second aspect the invention provides a murine skin organoid culture broth used in the method according to the first aspect.
Through the technical scheme, the invention at least has the following beneficial effects:
(1) The method provided by the invention adopts the murine skin tissue (or primary cells thereof) as the raw material, has wide sources, and also avoids the limitation of ethics caused by human experiment materials. And can select specific murine skin to prepare skin organoids according to different research needs, has the advantage of wide product applicability.
(2) The method provided by the invention has the advantages of simple, quick and efficient flow for preparing the murine skin organoids, and can effectively improve the experimental efficiency. In addition, in the method provided by the invention, the same culture solution is adopted for construction, passage and amplification of the murine skin organoids, so that the possibility of experimental errors caused by using different culture solutions at different stages in most of the existing organoid culture methods is reduced.
(3) The method provided by the invention has higher success rate when being used for preparing the murine skin organoids.
Drawings
FIG. 1 is a schematic diagram showing the morphology of the skin tissue organoids of the C57 mice prepared in example 1 (scale of 100 μm in the figure).
FIGS. 2-4 are schematic representations of the morphology of C57 mouse skin tissue organoids constructed using different murine skin organoid cultures in example 2 (scale 100 μm).
FIGS. 5 and 6 are schematic diagrams of morphology of C57 mouse skin tissue organoids constructed using mouse skin primary cells obtained by different digests in example 3 (scale of 100 μm in the figures), respectively.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The inventor of the present invention has skillfully found in the research that the murine skin organoids can be obtained by directly placing the murine skin primary cells in a specific medium for culture. Through further research, the inventor finds that when a specific small molecular substance is added into a basic culture medium (such as an Advanced DMEM/F12 culture medium and the like), the construction efficiency and success rate of constructing the skin organoid by the murine primary skin cells can be effectively improved. The culture medium can be used commonly in construction, expansion culture and passage of the murine skin organoids, so that the experimental process of construction of the murine skin organoids is greatly simplified, and experimental errors and material waste caused by misuse of the culture medium are reduced.
Based on this, in one aspect, the invention provides a method of constructing a murine skin organoid comprising placing murine skin primary cells in a murine skin organoid culture medium for organoid culture, wherein the murine skin organoid culture medium comprises nicotinamide, dexamethasone, and Y-27632.
Dexamethasone (DXMS) is a synthetic corticosteroid with CAS number 50-02-2.
Y-27632 is an RHO/ROCK pathway inhibitor, CAS number 146986-50-7, of formula (I) (usually supplied as the dihydrochloride salt).
According to a preferred embodiment of the present invention, wherein the composition of the murine skin organoid culture medium comprises a basal medium and additives, preferably the basal medium is Advanced DMEM/F12 medium.
Preferably, the additive comprises 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), L-glutamine supplement, N-2 additive, B-27 additive, N-acetylcysteine, R-spinal protein (R-Spondin, RSPO), epidermal Growth Factor (EGF), WNT protein, noggin (Noggin), fibroblast growth factor 10 (FGF 10), bovine Serum Albumin (BSA), A83-01, SB202190, forskolin (Forskolin), nicotinamide, dexamethasone, Y-27632, and antibiotics. Preferably, the antibiotic is at least one selected from the group consisting of penicillin, streptomycin and amphotericin B.
In the present invention, the L-glutamine supplement means a compound capable of providing L-glutamine to a medium, and any substance having the above-mentioned functions in the art can be applied to the present invention. For example, L-glutamine, L-alanyl-L-glutamine dipeptide can be used. The present invention is not particularly limited as to the specific source of the L-glutamine supplement, and may be either a self-prepared product according to the prior art or a related product directly obtained by commercial use (e.g., glutamax TM Etc.).
Both the N2 and B27 additives are serum-free additives used in the medium, which can provide the medium with a variety of nutrients and cytokines, and are generally available commercially. The N2 and B27 additives obtained commercially are usually supplied as concentrates (e.g., 100X or 50X) and may be diluted with medium or PBS prior to use or may be added directly to the medium at the desired concentration.
R-spinal proteins are a recently newly discovered secreted protein family, and 4 members (sequentially named RSPO1-RSPO 4) which bind LGR4 (Leucine-rich repeat-containing G protein-linked receptor 4, also called GPR 48) in the G-protein coupled receptor family and activate the Wnt/beta-catenin signaling pathway have been discovered. The present invention is not particularly limited as to the R-spinal protein used, and any R-spinal protein that can be used for (murine) skin organoid construction can be applied to the present invention, such as RSPO-1, RSPO-3, etc. (preferably RSPO-1).
WNT proteins are a highly conserved family of secreted glycoproteins, are important proteins of the Wnt signaling pathway, and can be added as cytokines to culture media for organoid construction. The present invention is not particularly limited as to the WNT proteins used, and any WNT proteins that can be used in (murine) dermal organoids construction are applicable to the present invention, such as WNT3a and the like.
A83-01 is an ALK5 inhibitor, with CAS number 909910-43-6, and the structural formula is shown as formula (II) below.
SB202190 is a p38 MAPK inhibitor with CAS number 152121-30-7 and structural formula shown in the following formula (III).
Forskolin is a medicine for treating heart disease, hypertension, diabetes and asthma, and can increase intracellular cAMP level, with CAS number 66575-29-9, and structural formula shown as formula (IV) below.
In the present invention, the amount of the additive to be used in the culture solution for murine skin organoids is not particularly limited, as long as it is capable of facilitating the construction of murine skin organoids.
In order to improve the construction efficiency and success rate of the murine skin organoids. According to a preferred embodiment of the present invention, wherein the amount of additive in the murine skin organoid culture medium is such that:
the final concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid is not lower than 5mM;
the amount of the L-glutamine supplement is such that the final concentration of L-glutamine in the culture broth is not less than 1mM;
the final concentration of the N-2 additive is not less than 0.5×;
the final concentration of the B-27 additive is not less than 0.5X;
the final concentration of N-acetylcysteine is not lower than 0.5mM;
the final concentration of R-spinal protein 1 is not lower than 50ng/mL;
the final concentration of the epidermal cell growth factor is not lower than 5ng/mL;
the final concentration of WNT protein is not lower than 5ng/mL;
the final concentration of Noggin is not lower than 10ng/mL;
the final concentration of fibroblast growth factor 10 is not lower than 5ng/mL;
the final concentration of bovine serum albumin is not less than 0.02 wt%;
the final concentration of A83-01 is not lower than 0.2 mu M;
the final concentration of SB202190 is not less than 5. Mu.M;
the final concentration of Forskolin is not lower than 5 mu M;
the final concentration of nicotinamide is not less than 5mM;
the final concentration of dexamethasone is not lower than 2 mu M;
the final concentration of Y27632 is not less than 5. Mu.M;
the final concentration of penicillin is not lower than 50U/mL;
the final concentration of streptomycin is not lower than 0.05mg/mL;
the final concentration of amphotericin B is not less than 0.2 μg/mL.
Preferably, in the murine skin organoid culture medium, the additives are used in an amount such that:
the final concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid is 8-12mM;
the amount of the L-glutamine supplement is such that the final concentration of L-glutamine in the culture broth is 2 to 3mM;
the final concentration of the N-2 additive is 1X-2X;
the final concentration of the B-27 additive is 1X-2X;
the final concentration of N-acetylcysteine is 1-1.5mM;
the final concentration of the R-spinal protein 1 is 100-500ng/mL;
the final concentration of the epidermal cell growth factor is 10-200ng/mL;
the final concentration of WNT protein is 10-500ng/mL;
the final concentration of Noggin is 50-200ng/mL;
the final concentration of the fibroblast growth factor 10 is 10-200ng/mL;
the final concentration of bovine serum albumin is 0.05-1 wt%;
the final concentration of A83-01 is 0.5-1 mu M;
the final concentration of SB202190 is 8-12. Mu.M;
the final concentration of Forskolin is 8-12 mu M;
the final concentration of nicotinamide is 8-12mM;
the final concentration of dexamethasone is 5-20 mu M;
the final concentration of Y27632 is 8-12 mu M;
the final concentration of penicillin is 100-200U/mL;
the final concentration of streptomycin is 0.1-0.2mg/mL;
the final concentration of amphotericin B is 0.25-0.5 μg/mL.
In the method provided by the invention, the source of the murine skin primary cells used is not particularly limited, and the murine skin primary cells can be obtained by self-preparation according to the prior art or can be related products obtained directly through commercial or custom-made.
According to a preferred embodiment of the invention, the method further comprises the step of preparing murine skin primary cells. Any means in the art of preparing primary cells from murine skin tissue may be suitable for use in this step of the invention. This step preferably comprises digesting the murine skin tissue fragments with a digestive juice a.
Preferably, this step further comprises sieving the tissue suspension obtained after digestion with a sieve having a pore size of 70-120 μm.
The digestive juice A used in the present invention has the function of resolving and dissociating murine skin tissues into single cells, and any substance or composition capable of achieving the object can be used as the digestive juice A in the method provided by the present invention.
Through a great deal of experiments, the inventor of the invention skillfully discovers that the digestion efficiency of the murine skin tissues can be further improved by combining specific digestive enzymes, and the activity of the obtained primary cells of the murine skin is ensured, so that the success rate and the efficiency of the subsequent construction of the murine skin organs are correspondingly improved.
According to a preferred embodiment of the invention, wherein the digestive juice a comprises collagenase IV, collagenase NB4 and deoxyribonuclease I.
Preferably, in the digestion liquid A, the content of collagenase IV is 50-500U/mL, the content of collagenase NB4 is 0.1-1PZ U/mL, and the content of deoxyribonuclease I is 0.05-0.2mg/mL.
In order to further increase the digestion efficiency of murine skin tissue while at the same time ensuring the activity of the murine skin primary cells, according to a preferred embodiment of the present invention, wherein the conditions of the digestion comprise: the volume ratio of the digestive juice A to the murine skin tissue fragments is 5-15:1, digestion temperature is 35-38 ℃ and digestion time is 40-80min. In order to bring the murine skin tissue fragments into sufficient contact with the digestive juice A, the digestion efficiency is improved, and local tissue over-digestion is avoided, preferably under shaking conditions of 100-200 rpm. In order to avoid degradation of activity or massive death of murine skin cells caused by overdigestion of murine skin tissue, it is preferable to observe the tissue digestion under a microscope every 5-15min during digestion. After more than 80% of the tissue is digested, the digestion can be stopped by adding PBS or medium.
In order to obtain a better digestion effect and to increase the yield of primary cells of murine skin obtained, it is preferable that the area of the pieces of murine skin tissue is not more than 3cm 2 . Preferably 1-2cm 2 。
According to a preferred embodiment of the invention, wherein the means for organoid culture comprises: the primary cells of the murine skin are coated by matrigel and then placed in a culture solution of the murine skin organ for culture.
The matrigel used in the present invention may be any matrigel used in the art that can be used in 3D culture of organoids or cells. It may be either a related product obtained by self-preparation according to the prior art or a related product obtained directly by commercial use (for example, matrigel manufactured by corning company, etc.).
Preferably, the conditions of the culture include: the temperature is 36-38 ℃, and the culture solution is replaced once in 3-5 days.
Preferably, the murine skin organoid culture medium is used in an amount of not more than 30% of the volume of the culture vessel. Preferably 15-25%. The volume of the culture vessel refers to the maximum volume of the culture vessel used in the construction of murine skin organoids, such as the volume of a flask, dish, or the volume of a single culture well in a culture plate. For example, when a 24-well cell culture plate is used, the murine skin organoid culture medium may be used in an amount of 600 to 800. Mu.L/well.
According to a preferred embodiment of the present invention, wherein the coating of murine skin primary cells with matrigel comprises: the murine skin primary cell suspension, cooled on ice, is mixed with matrigel and the suspension obtained is added to a culture vessel (for example a low-adsorption cell culture plate) and solidified at 36-38 ℃.
Preferably, the content of murine skin primary cells in said suspension is 1.2X10 4 -1.6×10 5 Individual cells/mL. Preferably 5X 10 4 -1.5×10 5 Individual cells/mL.
Preferably, the suspension is used in an amount of no more than 5% of the volume of the culture vessel. Preferably 1-3%. For example, when a 24 well cell culture plate is used, 50-100. Mu.L of suspension can be added per well.
More preferably, the volume ratio of added suspension to murine skin organoid culture broth is 1:8-12.
According to a preferred embodiment of the invention, wherein the method further comprises passaging murine skin organoids. Preferably, the passaging comprises the steps of organoid collection, organoid digestion, and continued culture after organoid passaging in sequence.
Preferably, the organoid collection method comprises removing the culture medium from the cell culture plate, adding pre-chilled buffer, placing the cell culture plate under cooling until the matrigel melts, and centrifuging to collect organoid pellet.
Preferably, the means of organoid digestion comprises mixing the collected organoid precipitate with digestive juice B.
More preferably, the digestive juice B contains trypsin or a pancreatin substitute, preferably a pancreatin substitute. In the present invention, pancreatin substitutes refer to other compounds, enzymes or compositions that have an action and effect similar to trypsin and that are capable of digesting tissue, but not trypsin. In the present invention, there are no particular restrictions on the source and type of the pancreatin substitutes, as long as they are capable of digesting organoid precipitates obtained by the method provided by the present invention, either as related products obtained by self-preparation or related products obtained by commercial or custom-made, such as TrypLE from ThermoFisher company TM Express, etc.
In the present invention, the amount of the digestive juice B is not particularly limited as long as the organoid precipitate can be efficiently digested. For example, when the pancreatin substitute TrypLE is used TM When Express is used as digestive juice B, the digestive juice B may be used in an amount of 1× for pancreatic enzyme substitutes relative to 1mL of organoid sediment.
Preferably, the means for continuing the culture after the organoid is passaged comprises: after coating the digested murine skin organoids with fresh matrigel, the following procedure was followed by 1:2-5 in ratio of wells to new cell culture plates, and placing in murine skin organoids culture medium for continuous culture. The "ratio of the number of wells" means the ratio of the number of wells in which the culture was inoculated into the culture plate before and after passage, for example, the culture inoculated into 1 culture well before passage was inoculated into 3 culture wells after passage, and the ratio of the number of wells was 1:3.
in a second aspect the invention provides a murine skin organoid culture broth for use in a method as described above. The characteristics of the culture solution are as described above and will not be described in detail herein.
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only and are not intended to limit the invention.
In the examples below, N2 additive was used as the additive, which was purchased from Thermo company, under the trade designation 17502048, B27 additive was purchased from Thermo company, under the trade designation 17504001, R-vertebra protein 1 was purchased from PeproTech company, under the trade designation 120-38-250UG, noggin was purchased from near shore protein company, under the trade designation CB89, A83-01 was purchased from MCE company, under the trade designation HY-10432, SB202190 was purchased from MCE company, under the trade designation HY-10295, Y-27632 was purchased from MCE company, and under the trade designation HY-10071. Unless specifically stated, the other reagents or consumables employed are all commercially available products from regular chemical/biological reagents or material suppliers, and the purity of the reagents is analytically pure.
Preparation example 1
This preparation is used to illustrate the composition and formulation of murine skin organoids medium and digest used in the examples below.
Preparation of culture Medium
The media for the construction of the skin organoids of mice were prepared according to the additive ingredients listed in Table 1, based on Advanced DMEM/F12.
TABLE 1
| Additive agent | Final concentration | Additive agent | Final concentration |
| HEPES | 10mM | FGF10 | 100ng/ml |
| L-glutamine | 2mM | BSA | 0.1％ |
| N2 additive | 1× | Nicotinamide | 1mM |
| B27 additive | 1× | A83-01 | 1μM |
| N-acetylcysteine | 1mM | SB202190 | 10μM |
| R-spondylin 1 | 250ng/ml | Dexamethasone | 10μM |
| EGF | 50ng/ml | Y-27632 | 10μM |
| Wnt-3a | 100ng/ml | Forskolin | 10μM |
| Noggin protein | 100ng/ml | Penicillin-streptomycin-amphotericin B | 1× |
* Penicillin-streptomycin-amphotericin B refers to a compound antibiotic prepared by mixing the three antibiotics, when the final concentration of penicillin-streptomycin-amphotericin B is 1 multiplied by 1, the final concentration of penicillin in a culture medium is 100U/mL, the final concentration of streptomycin is 0.1mg/mL, and the final concentration of amphotericin B is 0.25 mug/mL.
(II) preparation of digestive juice
Digestive juice was prepared according to the formulation of table 2.
TABLE 2
Example 1
This example is used to illustrate the effect of murine skin organoids constructed using the methods provided herein.
Skin tissue was taken after the neck of C57 mice was sacrificed, and primary and subculture of murine skin organoids was performed according to the following steps:
digestion and isolation of murine skin tissue
1. Hair and subcutaneous adipose tissue attached to the skin tissue of the mice were removed. The treated mouse skin tissue was then sheared into 1cm x 2cm pieces using sterile scissors and a scalpel.
2. The skin tissue fragments of the mice are completely transferred into a 15mL sterile centrifuge tube, and according to the amount of the tissue fragments, the skin tissue and digestive juice are 1:10 by volume, the digestion solution A of Table 2 was added, and the tube was then placed on a shaker with a constant temperature air bath and digested at 37℃with shaking at 200rpm for about 1 hour. Taking out every 5min in the middle, and observing the separation effect under a mirror. Epithelial cell debris was then scraped from the tissue mass with a blade, and undigested tissue debris was transferred to a new 15mL centrifuge tube and digestion was continued as described above. Finally, the cells obtained from the digestion were pooled and resuspended in murine skin organoids medium of Table 1.
3. The cell suspension obtained in step 2 was filtered with a 100 μm sieve and the cell filtrate was collected with a new 15mL centrifuge tube. Centrifuging the cell filtrate at 1000rpm for 5min, discarding supernatant, and preparing into about 1.5X10 with fresh murine skin organoid medium 5 The primary cell suspension of the murine skin tissue of individual cells/mL was placed on ice and cooled for 5min for use.
(II) Primary organoid culture
1. And (3) adding Matrigel (the volume ratio of the cell suspension to the Matrigel is about 1:2) into the cooled primary cell suspension of the murine skin tissue obtained in the step (one), and uniformly mixing.
2. The mixture of cells and Matrigel was dropped on a pre-heated 24 well low adsorption culture plate at about 70 μl per drop. The plates were placed in a 37℃incubator (5% CO) 2 ) Heating for 30min, taking out the culture plate after the colloid is solidified, and adding 700 mu L/hole of preheated murine skin organoid culture liquid. Proper amount of sterile PBS or double distilled water is added into the peripheral holes, so that the evaporation of the culture solution is reduced.
3. The culture condition of organoids is observed every day, and the culture solution is changed for 3-5 days according to the evaporation and color change condition of the culture medium. When the culture solution is added, the suction head faces to the side wall, so that the matrigel is prevented from being damaged.
(III) organoid subculture
When the organoids grow to a certain number and size (the diameter is usually about 500 μm), the organoids can be passaged. The specific operation steps are as follows:
1. the broth was removed, pre-chilled PBS was added and the matrigel was thawed (about 1 h) on ice. Then, the organoid pellet was collected by centrifugation at 1300rpm for 5min at 4 ℃.
2. Digestion solution B (about 800. Mu.L/well) in Table 2 was added to the organoid pellet, digested at 37℃and observed at 2min intervals during which time the digestion was stopped by adding medium after organoid had been digested to smaller clumps (less than 10 agglomerated cells in the cell mass).
3. After the digestion had been terminated, the mixture was centrifuged at 1300rpm at 4℃for 5min, the supernatant was discarded and the mixture was used as a sample according to 1:2-5 well count ratio by adding an appropriate amount of thawed fresh Matrigel (well count ratio was determined based on cell count results, about 1X 10 after passage) 5 Individual cells/well), and mixing. The cell-containing matrigel was inoculated into a new 24-well ultra-low adsorption plate at an amount of 70. Mu.L/well, heated at 37℃for 30min to coagulate the gel, and then 700. Mu.L/well of murine skin tissue organoid culture medium was added. Culturing the skin tissue organoids after passage according to the method in the step (two).
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein 6 holes are planted in total, and after 14 days of culture, the first passage is carried out, and at the moment, 6 holes are all successful in obtaining the skin tissue organoids of the mice (organoid morphology diagram is shown in figure 1). The observation shows that the skin tissue organoid of the mice at the first passage has better activity, and the organoid state is still good after 5 passages.
Example 2
This example is intended to illustrate the effect of the composition of the murine skin organoid culture medium on the murine skin organoid construction effect.
(1) The procedure of example 1 was followed except that the murine skin organoid culture broth used was free of nicotinamide, and the specific procedure and conditions were the same as in example 1.
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein 6 holes are planted in total, and after 14 days of culture, the first passage is carried out, 4 holes successfully obtain skin tissue organoids (organoid morphology diagram is shown in figure 2), 0 holes cell death, and 2 holes do not produce skin tissue organoids.
(2) The procedure of example 1 was followed, except that the amount of epidermal growth factor in the murine skin organoid medium used was adjusted to 5ng/mL, and the procedure and conditions were the same as in example 1.
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein 6 holes are planted in total, and after 14 days of culture, the first passage is carried out, 2 holes successfully obtain skin tissue organoids (the morphological diagram is shown in figure 3), 0 holes cell death, and 4 holes do not produce skin tissue organoids.
(3) The procedure of example 1 was followed except that the murine skin organoid medium used was replaced with R-vertebrate protein 3 in the R-vertebrate protein 1 fraction, and the procedure and conditions were the same as in example 1.
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein 6 holes are planted in total, and the first passage is carried out after the culture for 14 days, at the moment, 6 holes are all successful in obtaining skin tissue organoids (the morphological diagram is shown in figure 4).
As can be seen from a comparison of the results of example 2 and example 1, when the method provided by the present invention is used for constructing a murine skin tissue organoid, the formation of the skin organoid is affected by the absence of certain components (e.g., nicotinamide, etc.) in the culture solution or by adjusting the content of certain components (e.g., reducing the content of epidermal growth factor). While the type of replacement R-spinal protein does not affect organoid morphology.
Example 3
This example is intended to illustrate the effect of choice of digestive juice used to digest mouse skin tissue on the effects of murine skin organoids construction.
(1) The procedure of example 1 was followed, except that in step (I), collagenase A (at a concentration of 2.5 mg/mL) was used in place of the digestive juice A in Table 2 to digest the skin tissue of the mice. The rest of the operation and conditions were the same as in example 1.
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein, 6 holes are planted in total, and after 14 days of culture, the first passage is carried out, at this time, only 1 hole successfully obtains skin tissue organoids (the morphological diagram is shown in fig. 5), 0 hole cell death, and 5 holes do not produce skin tissue organoids.
(2) The procedure of example 1 was followed except that the amount ratio of each enzyme in the digestive juice A in Table 2 was adjusted (see Table 3 for details), and the skin tissue of the mice was digested with the adjusted first digestive juice. The rest of the operation and conditions were the same as in example 1.
TABLE 3 Table 3
The same batch of mouse skin tissue was treated by the method described above to construct mouse skin tissue organoids. Wherein, 6 holes are planted in total, and after 14 days of culture, the first passage is carried out, at this time, only 1 hole successfully obtains skin tissue organoids (the morphological diagram is shown in fig. 6), 0 hole cell death, and 5 holes do not produce skin tissue organoids.
As can be seen from comparison of the results of example 3 and example 1, when the choice of the digestive juice used for digesting the skin tissue of the mouse has a large influence on the construction of the skin tissue organoids of the mouse, the success rate of the construction of the skin tissue organoids of the mouse is significantly reduced when the composition of the digestive juice or the content thereof is deviated from the preferred range of the present invention.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A method of constructing a murine skin organoid, comprising placing murine skin primary cells in a murine skin organoid culture medium for organoid culture, wherein the murine skin organoid culture medium comprises nicotinamide, dexamethasone, and Y-27632.
2. The method of claim 1, wherein the components of the murine skin organoid culture medium comprise basal medium and additives, preferably the basal medium is Advanced DMEM/F12 medium;
preferably, the additive comprises 4-hydroxyethyl piperazine ethane sulfonic acid, an L-glutamine supplement, an N-2 additive, a B-27 additive, N-acetylcysteine, R-spinal protein, epidermal cell growth factor, WNT protein, noggin, fibroblast growth factor 10, bovine serum albumin, A83-01, SB202190, forskolin, nicotinamide, dexamethasone, Y-27632, and an antibiotic, preferably the antibiotic is selected from at least one of penicillin, streptomycin, and amphotericin B.
3. The method of claim 2, wherein the murine skin organoid culture medium is supplemented with an additive in an amount such that:
the final concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid is not lower than 5mM, preferably 8-12mM;
the amount of the L-glutamine supplement is such that the final concentration of L-glutamine in the culture broth is not less than 1mM, preferably 2 to 3mM;
the final concentration of the N-2 additive is not less than 0.5X, preferably 1X-2X;
the final concentration of the B-27 additive is not less than 0.5X, preferably 1X-2X;
the final concentration of N-acetylcysteine is not less than 0.5mM, preferably 1-1.5mM;
the final concentration of R-spinal protein is not lower than 50ng/mL, preferably 100-500ng/mL;
the final concentration of the epidermal growth factor is not lower than 5ng/mL, preferably 10-200ng/mL;
the final concentration of WNT protein is not less than 5ng/ml, preferably 10-500ng/ml;
the final concentration of noggin is not lower than 10ng/mL, preferably 50-200ng/mL;
the final concentration of the fibroblast growth factor 10 is not lower than 5ng/mL, preferably 10-200ng/mL;
the final concentration of bovine serum albumin is not less than 0.02 wt%, preferably 0.05-1 wt%;
the final concentration of A83-01 is not less than 0.2. Mu.M, preferably 0.5 to 1. Mu.M;
the final concentration of SB202190 is not less than 5. Mu.M, preferably 8-12. Mu.M;
the final concentration of Forskolin is not less than 5. Mu.M, preferably 8-12. Mu.M;
the final concentration of nicotinamide is not less than 5mM, preferably 8-12mM;
the final concentration of dexamethasone is not lower than 2. Mu.M, preferably 5-20. Mu.M;
the final concentration of Y27632 is not less than 5. Mu.M, preferably 8-12. Mu.M;
the final concentration of penicillin is not lower than 50U/mL, preferably 100-200U/mL;
the final concentration of streptomycin is not lower than 0.05mg/mL, preferably 0.1-0.2mg/mL;
the final concentration of amphotericin B is not lower than 0.2. Mu.g/mL, preferably 0.25-0.5. Mu.g/mL.
4. The method of claim 1, wherein the method further comprises the step of preparing murine skin primary cells; this step preferably comprises digesting the murine skin tissue fragments with a digestive juice a;
preferably, this step further comprises sieving the tissue suspension obtained after digestion with a sieve having a pore size of 70-120 μm.
5. The method of claim 4, wherein the digestive juice a comprises collagenase IV, collagenase NB4, and deoxyribonuclease I;
preferably, in the digestion liquid A, the content of collagenase IV is 50-500U/mL, the content of collagenase NB4 is 0.1-1PZ U/mL, and the content of deoxyribonuclease I is 0.05-0.2mg/mL.
6. The method of claim 4, wherein the conditions of digestion comprise: the volume ratio of the digestive juice A to the murine skin tissue fragments is 5-15:1, the digestion temperature is 35-38 ℃, the digestion time is 40-80min, and the digestion is preferably carried out under the shaking condition of 100-200 rpm;
preferably, the area of the murine skin tissue fragments does not exceed 3cm 2 Preferably 1-2cm 2 。
7. The method of claim 1, wherein the organoid is cultured in a manner comprising: coating the primary cells of the murine skin with matrigel, and then culturing in a murine skin organ culture medium;
preferably, the conditions of the culture include: the temperature is 36-38 ℃, and the culture solution is replaced for 3-5 days;
preferably, the murine skin organoid culture medium is used in an amount of not more than 30% of the volume of the culture vessel.
8. The method of claim 7, wherein coating the murine skin primary cells with matrigel comprises: mixing the murine skin primary cell suspension cooled on ice with matrigel, adding the obtained suspension into a culture vessel, and solidifying the suspension at 36-38deg.C;
preferably, the content of murine skin primary cells in said suspension is 1.2X10 4 -1.6×10 5 Individual cells/mL;
preferably, the suspension is used in an amount of not more than 5% of the volume of the culture vessel, preferably in a volume ratio of suspension to murine skin organoid culture broth of 1:8-12.
9. The method according to any one of claims 1-8, wherein the method further comprises passaging of murine skin organoids, preferably the passaging comprises the steps of organoid collection, organoid digestion, organoid passaging followed by further culturing;
preferably, the organoid collection method comprises removing the culture solution in the cell culture plate, adding pre-cooled buffer solution, placing the cell culture plate under cooling condition until matrigel is melted, and centrifuging to collect organoid precipitate;
preferably, the means of organoid digestion comprises mixing the collected organoid precipitate with digestive juice B;
more preferably, the digestive juice B contains trypsin or pancreatin substitutes, preferably pancreatin substitutes;
preferably, the means for continuing the culture after the organoid is passaged comprises: after coating the digested murine skin organoids with fresh matrigel, the following procedure was followed by 1:2-5 in ratio of wells to new cell culture plates, and placing in murine skin organoids culture medium for continuous culture.
10. A murine skin organoid culture medium used in the method of any of claims 1-9.
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