Middle ear organoid culture medium and culture method

Abstract

The application discloses a kind of middle ear organoids culture medium and culture method, belong to organoids technical field.The middle ear organoids culture medium includes: basic culture medium, specific additive factor, CHIR99021 and TTNPB;Specific additive factor includes the following end concentration components: SB203580, 1-20 mmol / L;SB431542, 50-1000 nmol / L;Y-27632, 1-20 mmol / L;FGF-10, 10-30 μg / L;RS-246204, 1-20 nmol / mL and the like.Culture method includes: middle ear tissue is handled to obtain middle ear cell, after middle ear cell is resuspended, with matrigel is mixed, then inoculation, after it coagulates, the above-mentioned middle ear organoids culture medium is added and is cultivated, obtains middle ear organoids.The culture medium of the application can simultaneously improve the quantity and survival rate of middle ear organoids, conducive to industrialized production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoids, in particular to a middle ear organoid culture medium and culture method. BACKGROUND

[0002] As an important model for studying hearing loss, infectious diseases and drug screening, middle ear organoids have become a research hotspot in the field of biomedicine in recent years. The related prior art solutions mainly include:

[0003] 1. Cochlea organoid model: a cochlea organoid with functional synapses is constructed through a staged 3D co-culture system, Wnt and YAP signaling pathways are used to activate cochlea progenitor cell proliferation, and functional connection is realized by combining nerve process growth.

[0004] 2. 3D bioprinting scaffold technology: a PVA / Gel / SA three-dimensional scaffold is used to construct a Corti organoid, which simulates the structure of the cochlea and is used for drug protection effect research.

[0005] 3. Construction method of inner ear organoids based on self-assembly: preparing a GelMA hydrogel; taking the inner ear basement membrane for dissection to obtain inner ear single cells; placing the inner ear single cells into a culture medium mixed with the GelMA hydrogel; and culturing the inner ear single cells to proliferate. The method of generating and amplifying the inner ear organoids by using the hydrogel substrate.

[0006] 4. Establishing inner ear organoids by inducing differentiation of human induced pluripotent stem cells: the method of culturing human induced pluripotent stem cells, inducing blastoids, differentiating blastoids, forming ear vesicles, and maturing inner ear organoids overcomes the shortcomings of the prior art. First, human induced pluripotent stem cells can be induced by dedifferentiation from human blood cells, skin cells, etc., solving the problem of cell source. In addition, due to the totipotency of pluripotent stem cells, the inner ear organoids differentiated from pluripotent stem cells contain a variety of cell components, including mechanically sensitive hair cells that can transmit sound, gravity and head movement signals. In addition, the scheme of generating organoids from pluripotent stem cells can edit and label the stem cells, and can be differentiated in large quantities and standardized, so it has broad application prospects.

[0007] However, there are few reports on middle ear organoids at present, and middle ear organoids are usually difficult to culture successfully and grow very slowly during the culture process. SUMMARY

[0008] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a middle ear organoid culture medium and culture method to solve the problems of low success rate, long culture period, poor survival ability and poor subculture ability of existing middle ear organoids during the culture process.

[0009] The technical scheme for solving the above technical problems is as follows: a middle ear organoid culture medium is provided, comprising: a basic culture medium, specific additives, CHIR99021 and TTNPB; wherein the specific additives comprise the following components at the following final concentrations: Glutamax, 0.5-2x; HEPES, 0.5-2x; N-Acetylcysteine, 0.5-2 mM; Nicotinamide, 1-20 mM; Penicillin streptomycin, 0.5-3x; B27, 0.5-2x; SB203580, 1-20 mmol / L; SB431542, 50-1000 nmol / L; Y-27632, 1-20 mmol / L; FGF-10, 10-30 μg / L; RS-246204, 1-20 nmol / mL; Noggin, 50-200 ng / mL; and EGF, 10-100 ng / mL.

[0010] On the basis of the above technical scheme, the application can also be improved as follows:

[0011] Further, the specific additives comprise the following components at the following final concentrations: Glutamax, 1x; HEPES, 1x; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1x; B27, 1x; SB203580, 10 mmol / L; SB431542, 500 nmol / L; Y-27632, 10 mmol / L; FGF-10, 20 μg / L; RS-246204, 10 nmol / mL; Noggin, 100 ng / mL; and EGF, 50 ng / mL.

[0012] Further, the final concentration of CHIR99021 in the middle ear organoid culture medium is 1-3 μg / L; preferably, the final concentration of CHIR99021 in the middle ear organoid culture medium is 2 μg / L.

[0013] Further, the final concentration of TTNPB in the middle ear organoid culture medium is 1-2 μg / L; preferably, the final concentration of TTNPB in the middle ear organoid culture medium is 2 μg / L.

[0014] Further, the basic culture medium is an Advanced DMEM / F-12 culture medium.

[0015] The method for culturing a middle ear organoid using the above middle ear organoid culture medium comprises the following steps:

[0016] The middle ear tissue is treated to obtain middle ear cells, the middle ear cells are mixed with Matrigel after resuspension, then inoculated, and after the Matrigel is solidified, the middle ear organoid culture medium is added for culture to obtain the middle ear organoid.

[0017] Further, the treatment of the middle ear tissue specifically comprises: taking the middle ear tissue, performing digestion treatment on the middle ear tissue with a digestion solution, then terminating the digestion, filtering, centrifuging, discarding the supernatant, and obtaining middle ear cells.

[0018] The more specific steps for treating the middle ear tissue are: taking the middle ear tissue, removing the muscle tissue around the middle ear tissue, then transferring the middle ear tissue to a pre-cooled container, adding a digestion solution, cutting the tissue into small pieces, then transferring the tissue pieces to another container, adding the digestion solution again for digestion, finally adding DPBS to terminate the digestion, filtering with a filter screen, centrifuging the filtered solution, discarding the supernatant, collecting the cell precipitate to obtain the middle ear cells.

[0019] Further, the middle ear tissue is a mammalian middle ear tissue, such as mouse middle ear tissue, human middle ear tissue, etc.

[0020] Further, the temperature during the digestion process is 35-40 DEG C, and the digestion time is 15-30 min.

[0021] Further, after the middle ear organoid culture medium is added, the culture is carried out at 37 DEG C under 5% CO2 conditions.

[0022] Further, the culture medium is replaced every 2-3 days during the culture process, and the culture is carried out for 4-8 days to obtain the middle ear organoid.

[0023] The present application has the following beneficial effects:

[0024] The present application simulates the mouse middle ear mucosa, fills the blank of the middle ear organoid model. The culture medium provided by the present application provides the reagent for activating Wnt3 signal and inhibiting MAPK signal required for the growth of the middle ear organoid. The present application has carried out in-depth research and development on the synergistic cooperation of the culture medium components, so that the components in the culture medium synergistically cooperate to play a role, which can improve the proliferation speed of the middle ear organoid, shorten the construction time of the middle ear organoid, improve the construction success rate of the middle ear organoid, and is beneficial to industrialized production; at the same time, the survival rate of the obtained middle ear organoid is high, that is, the number and survival rate of the middle ear organoid can be improved at the same time by using the middle ear organoid culture medium provided by the present application to culture the middle ear organoid. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a schematic diagram of the middle ear organoid culture of Example 1 under 4 times magnification at 0 day.

[0026] Figure 2 It is a schematic diagram of the middle ear organoid culture of Example 1 under 20 times magnification at 0 day.

[0027] Figure 3 Schematic of a 4x view of the ear organoids at 1 day of culture in Example 1.

[0028] Figure 4 Schematic of a 20x view of the ear organoids at 1 day of culture in Example 1.

[0029] Figure 5 Schematic of a 4x view of the ear organoids at 2 days of culture in Example 1.

[0030] Figure 6 Schematic of a 20x view of the ear organoids at 2 days of culture in Example 1.

[0031] Figure 7 Schematic of a 4x view of the ear organoids at 3 days of culture in Example 1.

[0032] Figure 8 Schematic of a 20x view of the ear organoids at 3 days of culture in Example 1.

[0033] Figure 9 Schematic of a 4x view of the ear organoids at 4 days of culture in Example 1.

[0034] Figure 10 Schematic of a 20x view of the ear organoids at 4 days of culture in Example 1.

[0035] Figure 11 Schematic of a 4x view of the solid sphere shaped organoids at 7 days of culture in Example 1.

[0036] Figure 12 Schematic of a 20x view of the solid sphere shaped organoids at 7 days of culture in Example 1.

[0037] Figure 13 Schematic of a 4x view of the vacuolated shaped organoids at 7 days of culture in Example 1.

[0038] Figure 14 Schematic of a 20x view of the vacuolated shaped organoids at 7 days of culture in Example 1.

[0039] Figure 15 Schematic of a 4x view of the vacuolated shaped organoids at 8 days of culture in Example 1.

[0040] Figure 16 Schematic of a 20x view of the vacuolated shaped organoids at 8 days of culture in Example 1.

[0041] Figure 17Schematic of solid sphere shaped organoids at 4x magnification at 8 days of culture of ear organoids in Example 1.

[0042] Figure 18 Schematic of solid sphere shaped organoids at 20x magnification at 8 days of culture of ear organoids in Example 1.

[0043] Figure 19 Schematic at 4x magnification at 8 days of culture of ear organoids in Example 2.

[0044] Figure 20 Schematic at 4x magnification at 8 days of culture of ear organoids in Example 3.

[0045] Figure 21 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 1.

[0046] Figure 22 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 2.

[0047] Figure 23 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 3.

[0048] Figure 24 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 4.

[0049] Figure 25 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 5.

[0050] Figure 26 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 6.

[0051] Figure 27 Schematic at 4x magnification at 8 days of culture of ear organoids in Comparative Example 7. DETAILED DESCRIPTION

[0052] The following examples are offered to illustrate the present application and are not intended to limit the scope of the present application. In the examples, unless otherwise indicated, conventional conditions or manufacturer's recommended conditions were employed. Where the reagent or instrument is not specifically indicated, a conventional product available from commercial suppliers was used.

[0053] Example 1:

[0054] A middle ear organoid culture medium comprises: a basic medium, specific additives, CHIR99021 and TTNPB; wherein the basic medium is an Advanced DMEM / F-12 medium; the specific additives comprise the following components at the following final concentrations: Glutamax, 1x; HEPES, 1x; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1x; B27, 1x; SB203580, 10 mmol / L; SB431542, 500 nmol / L; Y-27632, 10 mmol / L; FGF-10, 20 μg / L; RS-246204, 10 nmol / mL; Noggin, 100 ng / mL; and EGF, 50 ng / mL; the final concentration of CHIR99021 in the middle ear organoid culture medium is 2 μg / L, and the final concentration of TTNPB is 2 μg / L.

[0055] The method for culturing a middle ear organoid by using the above middle ear organoid culture medium comprises the following steps:

[0056] (1) After the mouse is executed by decapitation in a physical and chemical room, it is soaked in 75% ethanol, transferred from the transmission window to the intercellular space, and placed on an absorbent paper with the head cut off by scissors. The mouse middle ear is taken out with scissors and forceps. The mouse middle ear tissue is repeatedly rinsed in pre-cooled DPBS, and the surrounding muscle tissue is removed with forceps.

[0057] (2) The mouse middle ear tissue rinsed in step (1) is transferred to a 1.5 mL pre-cooled EP tube with forceps, and 200 μL of digestion solution I (MasterAim ® tissue digestion solution, 10-100-046) is added. Then it is manually cut into small pieces of about 1 mm. The tissue pieces are all transferred to a 15 mL centrifuge tube, 4 mL of digestion solution I is added, and it is shaken and digested in a 37℃ incubator for 30 min. During this period, the cell leakage is observed under a microscope.

[0058] (3) If the red blood cell sedimentation is obvious, add red blood cell lysis solution to resuspend the cell sediment, and lyse at room temperature for 5 min. After stopping the digestion by adding 2 times the volume of DPBS, the cell suspension is passed through a 100 μm filter screen pre-washed with DPBS, and the filtrate is collected at room temperature and centrifuged at 430 g for 5 min. The supernatant is discarded and the cell sediment is collected.

[0059] (4) Resuspend the cell pellet collected in step (3) with the above-mentioned middle ear organoid culture medium, then add 2 times volume of undiluted Matrigel (Corning, 356231) and mix carefully by pipetting up and down 10 times to fully mix, avoiding air bubbles during the process.

[0060] (5) Take 25 μL of the cell suspension and add to the center of each well of a pre-warmed 48-well plate, so that the sample forms a dome structure in the center of each well. In order to prevent air bubbles from occurring during inoculation, do not completely expel the liquid in the gun tip.

[0061] (6) Place the inoculated culture dish in a CO2 incubator at 37°C for 5 min, and after the gel droplet does not flow obviously after gentle shaking, carefully invert it and allow it to fully solidify for 25 min. Add 200 μL of middle ear organoid culture medium, and after microscopic examination and photographing, place it in the incubator at 37°C, 5% CO2.

[0062] (8) Observe daily, replace the new culture medium every 2 days during the period, and take photographs under a microscope after 8 days of culture.

[0063] Example 2:

[0064] A middle ear organoid culture medium comprises: a basic culture medium, specific additives, CHIR99021 and TTNPB; wherein the basic culture medium is Advanced DMEM / F-12 culture medium; the specific additives comprise the following components at the following final concentrations: Glutamax, 0.5x; HEPES, 0.5x; N-Acetylcysteine, 0.5 mM; Nicotinamide, 1 mM; Penicillin streptomycin, 0.5x; B27, 0.5x; SB203580, 1 mmol / L; SB431542, 50 nmol / L; Y-27632, 1 mmol / L; FGF-10, 10 μg / L; RS-246204, 1 nmol / mL; Noggin, 50 ng / mL; and EGF, 10 ng / mL; the final concentration of CHIR99021 in the middle ear organoid culture medium is 1 μg / L, and the final concentration of TTNPB is 1.5 μg / L.

[0065] The method for culturing a middle ear organoid using the above-mentioned middle ear organoid culture medium is the same as that in Example 1.

[0066] Example 3:

[0067] A middle ear organoid culture medium comprises: a basic medium, specific additives, CHIR99021 and TTNPB; wherein the basic medium is an Advanced DMEM / F-12 medium; the specific additives comprise the following components at the following final concentrations: Glutamax, 2x; HEPES, 2x; N-Acetylcysteine, 2 mM; Nicotinamide, 20 mM; Penicillin streptomycin, 3x; B27, 2x; SB203580, 20 mmol / L; SB431542, 1000 nmol / L; Y-27632, 20 mmol / L; FGF-10, 30 μg / L; RS-246204, 20 nmol / mL; Noggin, 200 ng / mL; and EGF, 100 ng / mL; the final concentration of CHIR99021 in the middle ear organoid culture medium is 3 μg / L, and the final concentration of TTNPB is 2 μg / L.

[0068] The method for culturing the middle ear organoid using the above-mentioned middle ear organoid culture medium is the same as that in Embodiment 1.

[0069] Comparative Example 1

[0070] Comparative Example 1 differs from Embodiment 1 in that CHIR99021 is removed from the culture medium, and the rest is the same as Embodiment 1.

[0071] Comparative Example 2

[0072] Comparative Example 2 differs from Embodiment 1 in that TTNPB is removed from the culture medium, and the rest is the same as Embodiment 1.

[0073] Comparative Example 3

[0074] Comparative Example 3 differs from Embodiment 1 in that CHIR99021 and TTNPB are removed from the culture medium, and the rest is the same as Embodiment 1.

[0075] Comparative Example 4

[0076] Comparative Example 4 differs from Embodiment 1 in that CHIR99021 in the culture medium is replaced with Wnt3A recombinant protein, and the rest is the same as Embodiment 1.

[0077] Comparative Example 5

[0078] Comparative Example 5 differs from Embodiment 1 in that SB203580 is removed from the culture medium, and the rest is the same as Embodiment 1.

[0079] Comparative Example 6

[0080] The difference between Comparative Example 6 and Example 1 is that SB431542 was removed from the culture medium, while the rest was the same as in Example 1.

[0081] Comparative Example 7:

[0082] The difference between Comparative Example 7 and Example 1 is that SB203580 and SB431542 were removed from the culture medium, while the rest were the same as in Example 1.

[0083] Experimental example:

[0084] 1. The middle ear organoids obtained after 8 days of culture in Examples 1-3 and Comparative Examples 1-7 were observed, and the results are as follows: Figures 1-27 .

[0085] Depend on Figures 1-27 It can be seen that the middle ear organoids obtained by culturing using the culture medium of this invention began to form from the second day and gradually increased in size. The middle ear organoids obtained by this invention are numerous and have good activity. In contrast, the organoids formed in the comparative example are fewer in number and have poorer activity.

[0086] 2. Detection of the number and viability of middle ear organoids

[0087] Mouse middle ear organoids were cultured for 8 days using the culture media described in Examples 1-3 and Comparative Examples 1-7. The number and viability of the formed organoids are shown in Table 1.

[0088] The number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7 are shown in Table 1:

[0089] Table 1. Number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7

[0090]

[0091] As shown in Table 1, when CHIR99021 was removed from the culture medium, the growth of middle ear organoids was slow, the number of middle ear organoids was less than that in Examples 1-3, and their activity was poor.

[0092] When TTNPB was removed from the culture medium, the growth rate of the middle ear organoids slowed down, the diameter of the middle ear organoids was smaller, the number was less than in Examples 1-3, and the activity was poor.

[0093] When CHIR99021 and TTNPB were removed from the culture medium, the number of middle ear organoids formed was significantly reduced and the viability was lower compared to Examples 1-3, proving that CHIR99021 and TTNPB are key components in the culture medium for mouse middle ear organoids.

[0094] When CHIR99021 in the culture medium was replaced by Wnt3A recombinant protein, the number of middle ear organoids and the viability of middle ear organoids were significantly reduced compared with Examples 1-3, and after the above component was replaced, the cells grew slowly.

[0095] When SB203580 was removed from the culture medium, the number and viability of middle ear organoids were significantly reduced.

[0096] When SB431542 was removed from the culture medium, the number and viability of middle ear organoids were significantly reduced.

[0097] When SB203580 and SB431542 were removed from the culture medium at the same time, the number and viability of middle ear organoids were significantly reduced.

[0098] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A middle ear organoid culture medium, characterized by, comprises: a basal medium, specific factors, CHIR99021 and TTNPB; wherein the specific factors consist of the following components at the final concentrations: Glutamax, 0.5-2x; HEPES, 0.5-2x; N-Acetylcysteine, 0.5-2 mM; Nicotinamide, 1-20 mM; Penicillin streptomycin, 0.5-3x; B27, 0.5-2x; SB203580, 1-20 mmol / L; SB431542, 50-1000 nmol / L; Y-27632, 1-20 mmol / L; FGF-10, 10-30 μg / L; RS-246204, 1-20 nmol / mL; Noggin, 50-200 ng / mL and EGF, 10-100 ng / mL; the final concentration of CHIR99021 in the middle ear organoid culture medium is 1-3 μg / L; the final concentration of TTNPB is 1-2 μg / L.

2. The middle ear organoid culture of claim 1, wherein, the specific factors consist of the following components at the final concentrations: Glutamax, 1x; HEPES, 1x; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1x; B27, 1x; SB203580, 10 mmol / L; SB431542, 500 nmol / L; Y-27632, 10 mmol / L; FGF-10, 20 μg / L; RS-246204, 10 nmol / mL; Noggin, 100 ng / mL and EGF, 50 ng / mL.

3. The middle ear organoid culture of claim 1, wherein, the final concentration of CHIR99021 in the middle ear organoid culture medium is 2 μg / L.

4. The middle ear organoid culture medium of claim 1, wherein, the final concentration of TTNPB in the middle ear organoid culture medium is 2 μg / L.

5. The middle ear organoid culture medium of claim 1, wherein, the basal medium is Advanced DMEM / F-12 medium.

6. A method of culturing a middle ear organoid using the middle ear organoid culture medium of any one of claims 1-5, characterized in that, comprises the following steps: the middle ear tissue is treated to obtain middle ear cells, the middle ear cells are mixed with Matrigel after resuspension, then inoculated, and after the Matrigel solidifies, the above-mentioned middle ear organoid culture medium is added for culture to obtain middle ear organoids.

7. The method of claim 6, wherein, after adding the middle ear organoid culture medium, culture at 37℃, 5% CO2.

8. The method of claim 6, wherein, the culture medium is replaced every 2-3 days during the culture process, and the culture is carried out for 4-8 days to obtain middle ear organoids.

Images