Medium medium ear organoid culture medium and culture method

Abstract

The invention discloses a medium ear organoid culture medium and a culture method, and belongs to the technical field of organoids. The medium ear organoid culture medium comprises a basic culture medium, a specific addition factor, CHIR99021 and TTNPB, wherein the specific addition factor comprises the following components with the final concentration: SB203580, 1-20 mmol / L, SB203580, 1-20 mmol / L, the concentration of SB431542 is 50 to 1000 nmol / L; y-27632, the concentration of which is 1 to 20 mmol / L; 10 to 30 [mu] g / L of FGF-10; the molecular weight of the molecular sieve is 1-20 nmol / mL, RS-246204, 1-20 The culture method comprises the following steps: treating middle ear tissues to obtain middle ear cells, re-suspending the middle ear cells, mixing with matrigel, inoculating, and adding into the middle ear organ culture medium for culturing after the middle ear cells are solidified to obtain the middle ear organs. The culture medium can improve the number and motility rate of middle ear organs at the same time, and is beneficial to industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to a culture medium and a culture method for middle ear organoids. Background Art

[0002] Middle ear organoids, as an important model for studying hearing loss, infectious diseases, and drug screening, have become a research hotspot in the biomedical field in recent years. Existing technologies related to them mainly include: 1. Cochlear organoid model: Cochlear organoids with functional synapses were constructed through a staged 3D co-culture system. The Wnt and YAP signaling pathways were used to activate the proliferation of cochlear progenitor cells, and functional connections were achieved in combination with neurite growth.

[0003] 2. 3D bioprinting scaffold technology: Use PVA / Gel / SA three-dimensional scaffolds to construct Corti organoids, simulate the cochlear structure and be used for drug protection effect research.

[0004] 3. Method for constructing inner ear organoids based on self-assembly: Prepare GelMA hydrogel; Dissect the inner ear basilar membrane to obtain inner ear single cells; Place the inner ear single cells in a culture matrix mixed with the GelMA hydrogel; Proliferate and culture the inner ear single cells. This method uses hydrogel substrates to generate and expand inner ear organoids.

[0005] 4. Establishment of inner ear organoids through induced differentiation of human induced pluripotent stem cells: The method of utilizing human induced pluripotent stem cell culture, embryoid body induction, embryoid body differentiation, otic vesicle formation stage and inner ear organoid maturation overcomes the shortcomings of existing technologies. First, human induced pluripotent stem cells can be induced by dedifferentiation of human blood cells, skin cells, etc., which solves the problem of cell source; in addition, due to the totipotency of pluripotent stem cells, inner ear organoids differentiated from pluripotent stem cells contain a variety of cellular 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 allows for gene editing and labeling of stem cells, and can be differentiated in large quantities and in a standardized manner, so it has broad application prospects.

[0006] However, there are currently few reports on the research of middle ear organoids. Middle ear organoids are usually difficult to culture successfully and grow very slowly during the culture process. Summary of the Invention

[0007] In order to address the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a culture medium and culture method for middle ear organoids, so as to solve the problems of low culture success rate, long culture cycle, and poor survival and passage ability of existing middle ear organoids during culture.

[0008] 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.

[0009] On the basis of the above technical scheme, the application can also be improved as follows: 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.

[0010] 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.

[0011] 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.

[0012] Further, the basic culture medium is an Advanced DMEM / F-12 culture medium.

[0013] The method for culturing a middle ear organoid using the above middle ear organoid culture medium 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 is solidified, the middle ear organoid culture medium is added for culture, and the middle ear organoid is obtained.

[0014] Further, the treatment of the middle ear tissue is specifically as follows: the middle ear tissue is taken, subjected to digestion treatment with a digestion solution, then the digestion is terminated, filtered, centrifuged, the supernatant is discarded, and the middle ear cells are obtained.

[0015] The more specific steps for treating the middle ear tissue are as follows: the middle ear tissue is taken, and the muscle tissue around the middle ear tissue is removed, then transferred to a pre-cooled container, a digestion solution is added, the tissue is cut into small pieces, then the tissue pieces are transferred to another container, the digestion solution is added again for digestion, finally the DPBS is added to terminate the digestion, the filter screen is used for filtering, the filtered solution is collected for centrifugation, the supernatant is discarded, the cell precipitate is collected to obtain the middle ear cells.

[0016] Further, the middle ear tissue is a mammalian middle ear tissue, such as mouse middle ear tissue, human middle ear tissue, etc.

[0017] Further, the temperature during the digestion is 35-40 DEG C, and the digestion time is 15-30 min.

[0018] Further, after the middle ear organoid culture medium is added, the culture is carried out at 37 DEG C under the condition of 5% CO2.

[0019] Further, the culture medium is replaced every 2-3 days during the culture, and the culture is carried out for 4-8 days to obtain the middle ear organoid.

[0020] The present application has the following beneficial effects: 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

[0021] Figure 1 It is a schematic diagram of the middle ear organoid culture of Example 1 under 4 times magnification at 0 day.

[0022] Figure 2 It is a schematic diagram of the middle ear organoid culture of Example 1 under 20 times magnification at 0 day.

[0023] Figure 3 Schematic of 4X magnification of ear organoids at 1 day of culture in Example 1.

[0024] Figure 4 Schematic of 20X magnification of ear organoids at 1 day of culture in Example 1.

[0025] Figure 5 Schematic of 4X magnification of ear organoids at 2 days of culture in Example 1.

[0026] Figure 6 Schematic of 20X magnification of ear organoids at 2 days of culture in Example 1.

[0027] Figure 7 Schematic of 4X magnification of ear organoids at 3 days of culture in Example 1.

[0028] Figure 8 Schematic of 20X magnification of ear organoids at 3 days of culture in Example 1.

[0029] Figure 9 Schematic of 4X magnification of ear organoids at 4 days of culture in Example 1.

[0030] Figure 10 Schematic of 20X magnification of ear organoids at 4 days of culture in Example 1.

[0031] Figure 11 Schematic of solid sphere morphology organoids at 4X magnification at 7 days of culture in Example 1.

[0032] Figure 12 Schematic of solid sphere morphology organoids at 20X magnification at 7 days of culture in Example 1.

[0033] Figure 13 Schematic of vacuolated morphology organoids at 4X magnification at 7 days of culture in Example 1.

[0034] Figure 14 Schematic of vacuolated morphology organoids at 20X magnification at 7 days of culture in Example 1.

[0035] Figure 15 Schematic of vacuolated morphology organoids at 4X magnification at 8 days of culture in Example 1.

[0036] Figure 16 Schematic of vacuolated morphology organoids at 20X magnification at 8 days of culture in Example 1.

[0037] Figure 17 Schematic of solid sphere morphology organoids at 4X magnification at 8 days of culture in Example 1.

[0038] Figure 18 Schematic representation of solid sphere shaped organoids at 20x magnification at 8 days of culture of ear organoids in Example 1.

[0039] Figure 19 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Example 2.

[0040] Figure 20 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Example 3.

[0041] Figure 21 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 1.

[0042] Figure 22 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 2.

[0043] Figure 23 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 3.

[0044] Figure 24 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 4.

[0045] Figure 25 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 5.

[0046] Figure 26 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 6.

[0047] Figure 27 Schematic representation at 4x magnification at 8 days of culture of ear organoids in Comparative Example 7. DETAILED DESCRIPTION

[0048] 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 commercially was used.

[0049] Example 1: 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.

[0050] The method for culturing a middle ear organoid by using the above-mentioned middle ear organoid culture medium comprises the following steps: (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 skin of the head is cut open, and the two middle ears of the mouse are taken out with scissors and tweezers. The mouse middle ear tissue is repeatedly rinsed in pre-cooled DPBS, and the surrounding muscle tissue is removed with tweezers.

[0051] (2) The mouse middle ear tissue rinsed in step (1) is transferred to a 1.5 mL pre-cooled EP tube with tweezers, 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°C incubator for 30 min. During this period, the cell leakage is observed under a microscope.

[0052] (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 digestion, add 2 times the volume of DPBS to terminate the digestion, and then pass the cell suspension through a 100 μm filter screen pre-washed with DPBS. The filtrate is collected and centrifuged at 430 g for 5 min at room temperature. The supernatant is discarded and the cell sediment is collected.

[0053] (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.

[0054] (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.

[0055] (6) Place the inoculated culture dish in a CO2 incubator at 37℃ 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℃, 5% CO2.

[0056] (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.

[0057] Example 2: A middle ear organoid culture medium, comprising: 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.

[0058] The method for culturing a middle ear organoid using the above-mentioned middle ear organoid culture medium is the same as in Example 1.

[0059] Example 3: 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.

[0060] 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.

[0061] Comparative Example 1 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.

[0062] Comparative Example 2 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.

[0063] Comparative Example 3 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.

[0064] Comparative Example 4 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.

[0065] Comparative Example 5 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.

[0066] Comparative Example 6 Comparative Example 6 differs from Embodiment 1 in that SB431542 is removed from the culture medium, and the rest is the same as Embodiment 1.

[0067] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that SB203580 and SB431542 are removed from the culture medium, and the rest is the same as Example 1.

[0068] Test example: 1. The middle ear organoids obtained after culturing for 8 days in Examples 1-3 and Comparative Examples 1-7 were observed. Figures 1-27 .

[0069] Depend on Figures 1-27 As can be seen, the middle ear organoids cultured using the medium of the present invention began to form from the second day and gradually increased in size. The middle ear organoids obtained by the present invention were numerous and had good activity. In contrast, the organoids formed in the control sample were fewer and had poorer activity.

[0070] 2. Detection of the number and viability of middle ear organoids The culture medium of Examples 1-3 and Comparative Examples 1-7 was used to culture mouse middle ear organoids for 8 days. The number and viability of the organoids formed are shown in Table 1: The number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7 are shown in Table 1: Table 1 Number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7

[0071] As shown in Table 1, when CHIR99021 was removed from the culture medium, the middle ear organoids grew slowly, the number of middle ear organoids was less than that in Examples 1-3, and the activity was poor.

[0072] 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 small, the number was less than that in Examples 1-3, and the activity was poor.

[0073] When CHIR99021 and TTNPB were removed from the culture medium at the same time, the number of middle ear organoids formed was significantly reduced compared to Examples 1-3, and the viability was lower, proving that CHIR99021 and TTNPB are key components in the mouse middle ear organoid culture medium.

[0074] When CHIR99021 in the culture medium was replaced with Wnt3A recombinant protein, compared with Examples 1-3, the number of middle ear organoids formed and the viability of the middle ear organoids decreased significantly, and after replacing the above components, the cells grew slowly.

[0075] When SB203580 was removed from the culture medium, the number and viability of middle ear organoids decreased significantly.

[0076] When SB431542 was removed from the culture medium, the number and viability of middle ear organoids decreased significantly.

[0077] The number and viability of the middle ear organoids were significantly reduced when SB203580 and SB431542 were removed from the culture medium at the same time.

[0078] The above description is merely preferred embodiments of the present application, and not intended to limit the present application. Any modification, equivalent replacement and improvement made without departing from the spirit and principle of the present application shall fall within the scope of protection of the present application.

Claims

1. A middle ear organoid culture medium, characterized in that include: Basal medium, specific supplemental factors, CHIR99021, and TTNPB; wherein the specific supplemental factors include the following components at the following final concentrations: Glutamax, 0.5-2×; HEPES, 0.5-2×; N-Acetylcysteine, 0.5-2 mM; Nicotinamide, 1-20 mM; Penicillinstreptomycin, 0.5-3×; B27, 0.5-2×; 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.

2. The middle ear organoid culture medium according to claim 1, characterized in that Specific factors added included the following components at the following final concentrations: Glutamax, 1×; HEPES, 1×; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1×; B27, 1×; 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 medium according to claim 1, characterized in that The final concentration of CHIR99021 in the middle ear organoid culture medium is 1-3 μg / L.

4. The middle ear organoid culture medium according to claim 3, characterized in that The final concentration of CHIR99021 in the middle ear organoid culture medium was 2 μg / L.

5. The middle ear organoid culture medium according to claim 1, characterized in that The final concentration of TTNPB in the middle ear organoid culture medium is 1-2 μg / L.

6. The middle ear organoid culture medium according to claim 5, characterized in that The final concentration of TTNPB in the middle ear organoid culture medium was 2 μg / L.

7. The middle ear organoid culture medium according to claim 1, characterized in that The basic culture medium was Advanced DMEM / F-12 medium.

8. A method for culturing middle ear organoids using the middle ear organoid culture medium according to any one of claims 1 to 7, characterized in that: The following steps are involved: The middle ear tissue is processed to obtain middle ear cells, which are resuspended and mixed with matrigel, and then inoculated. After solidification, the above-mentioned middle ear organoid culture medium is added for culture to obtain middle ear organoids.

9. The method according to claim 8, characterized in that After adding the middle ear organoid culture medium, culture was carried out at 37°C and 5% CO2.

10. The method according to claim 8, characterized in that During the culture process, the culture medium was replaced every 2-3 days, and the middle ear organoids were obtained after 4-8 days of culture.

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